Modified adenoviral fiber with ablated to cellular receptors

ABSTRACT

The present invention concerns a modified adenoviral fiber containing at least one mutation affecting one or more amino acid residue(s) of said adenoviral fiber interacting with at least one glycosaminoglycan and/or sialic acid-containing cellular receptor, as well as a trimer of such a modified adenoviral fiber. The present invention also relates to a DNA fragment, an expression vector encoding said modified adenoviral fiber. The present invention also concerns an adenoviral particle lacking a wild-type fiber and comprising the trimer of modified adenoviral fibers as well as a process for producing such an adenoviral particle. Theto present invention also provides a composition comprising such an adenoviral particle and the therapeutic use thereof.

The present invention relates to an adenoviral fiber protein mutated inthe region(s) or residue(s) involved in recognizing and/or binding to atleast one cell-surface glycosaminoglycan or sialic acid-containingreceptor. It also relates to an adenovirus particle bearing at itssurface such a mutated fiber, having a reduced or ablated capacity tointeract with such glycosaminoglycan or sialic acid-containingreceptors. The present invention also provides an adenoviral fiberprotein mutated in the region(s) or residue(s) involved in recognizingand binding to both such glycosaminoglycan or sialic acid-containingreceptors and to the coxsackie-adenovirus receptor (CAR). It alsorelates to an adenovirus particle bearing at its surface such a doublymutated fiber, having a reduced or ablated capacity to interact withboth CAR and such glycosaminoglycan and/or sialic acid-containingcellular receptors. Such adenovirus particles can optionally be combinedwith a ligand which confers modified or retargeted host specificity. Theinvention is of most particular value in the context of adenovirustargeting and the development of targeted vectors that can be used formultiple gene therapy applications, including cancer, cardiovascular,genetic, and inflammatory diseases.

Adenoviruses have, been detected in many animal species, arenon-integrative and low pathogene. They are able to infect a variety ofcell types, dividing as well as quiescent cells. They have a naturaltropism for airway epithelia. In addition, they have been used as liveenteric vaccines for many years with an excellent safety profile.Finally, they can be easily grown and purified in large quantities.These features have made recombinant adenoviruses particularlyappropriate for use as gene therapy vectors for a large variety oftherapeutic and vaccine applications.

Adenoviral genome consists of a linear double-standed DNA molecule ofapproximately 36 kb (conventionally divided into 100 map units (mu))carrying more than about thirty genes necessary to complete the viralcycle. During productive adenoviral infection, three classes of viralgenes are temporally expressed in the following order: early (E),intermediate and late (L). The early genes are divided into 4 regionsdispersed in the adenoviral genome (E1 to E4). The E1, E2 and E4 regionsbeing essential to viral replication whereas E3 region is dispensable inthis respect. The E1 region (E1A and E1B) encodes proteins responsiblefor the regulation of transcription of the viral genome. Expression ofthe E2 region genes (E2A and E2B) leads to the synthesis of thepolypeptides needed for viral replication (Pettersson and Roberts, 1986,In Cancer Cells (Vol 4): DNA Tumor Viruses, Botchan and Glodzicker SharpEds pp 37-47, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).The proteins encoded by the E3 region prevent cytolysis by cytotoxic Tcells and tumor necrosis factor (Wold and Gooding, 1991, Virology 184,1-8). The proteins encoded by the E4 region are involved in DNAreplication, late gene expression, splicing and host cell shut off(Halbert et al., 1985, J. virol. 56, 250-257). The late genes (L1 to L5)are mostly transcribed from the major late promoter (MLP). They overlapat least in part with the early transcription units and encode in theirmajority the structural proteins constituting the viral capsid. Inaddition, the adenoviral genome carries at both extremities cis-actingregions essential for DNA replication, respectively the 5′ and 3′ ITRs(Inverted Terminal Repeats) which harbor origins of DNA replication andthe packaging sequence immediately adjacent to the 5′ITR.

Most of the adenoviral vectors presently used in gene therapy protocolsare replication-defective viruses (i.e. incapable of dividing orproliferating in the host cells they infect), to avoid theirdissemination in the environment and the host organism. The feasabilityof gene transfer using E1-deleted vectors has been demonstrated into avariety of tissues in vivo (see for example Yei et al., 1994, Hum. GeneTher. 5, 731-744; Dai et al., 1995, Proc. Natl. Acad. Sci. USA 92,1401-1405; Howell et al., 1998, Hum. Gene Ther. 9, 629-634; Nielsen etal., 1998, Hum. Gene Ther. 9, 681-694). However, their use is associatedwith acute inflammation and toxicity in a number of animal models (Yanget al., 1994, Proc. Natl. Acad. Sci. USA 91, 4407-4411; Zsengeller etal., 1995, Hum. Gene Ther. 6, 457-467) as well as with host immuneresponses to the viral vector and gene products (Yang et al., 1995, J.Virol. 69, 2004-2015), resulting in the elimination of the infectedcells and transient gene expression. Second-generation adenovirusvectors having additional viral genes deleted to overcomeadenovirus-mediated immunogenicity are currently investigated(Engelhardt et al., 1994, Hum. Gene Ther. 5, 1217-1229; Engelhardt etal., 1994, Proc. Natl. Acad. Sci. USA 91, 6196-6200). Evaluation of E1and partially E4-deleted adenoviral vectors in vivo have shown a reducedhepatotoxicity and inflammation (Christ et al., 2000, Human Gene Ther.11, 415-427).

The initial attachment of the adenovirus particle to the cell surface ismediated by the binding of the knob region of the viral fiber protein toubiquitous cell surface receptors. Two distinct proteins belonging tothe immunoglobulin superfamily were reported as the primary receptorsfor adenovirus serotype C fibers: the coxsackievirus-adenovirus receptor(termed CAR) (Bergelson et al, 1997, Science 275, 1320-1323. Tomko etal., 1997, Proc. Natl. Acad. Sci. USA 94, 3352-3356) and the alpha 2domain of the major histocompatibility complex class I molecule (Hong elal., 1997, EMBO J. 16, 2294-2306). A predominant role for CAR inadenovirus tropism is however suggested by the work of McDonald et al.(1999, Gene Ther. 6, 1512-1519), who demonstrated discordance betweenMHC class I heavy chain levels at the cell surface and adenovirussusceptibility. In addition to subgroup C adenoviral fibers, CAR wasalso shown to bind to subgroups A, D, E and F fibers (Roelvink et al.,1998, J. Virol. 72, 7909-7915) but not to subgroup B adenoviral fibers,such as those of serotype 3 and 7 (Krasnykh et al., 1996, J. Virol. 70,6839-6846; Santis et al., J. Gen Virol. 80, 1519-1527).

More recently, cell-surface heparan sulfate glycosaminoglycans (HSG)were shown to interact with adenovin's serotype 5 (Ad5), which suggeststhat these molecules may also facilitate virus binding to cells(Decheccili et al., 2000, Virology 268, 382-390; Dechecchi et al., 2001,J. Virol. 75, 8772-8780).

Internalization into the cell of the attached adenoviral particles ismediated by the recognition of the Arg-Gly-Asp (RGD) sequence located inthe viral penton base protein by the cellular alphav integrins (Mathiaset al., 1994, J. Virol. 68, 6811-6814). This interaction triggerscellular internalization whereby: the virions achieve localizationwithin the endosome. Acidification of the endosome elicits conformationchanges in the capsid proteins, allowing their interaction with theendosome membrane in a manner that achieves vesicle disruption andparticle escape. Following endosomolysis, the virion translocates to thenucleus, where the subsequent steps of the viral life cycle occur.

The almost ubiquitous distribution of the CAR cellular receptor isthought to be primarily responsible for the broad cell tropism of thehuman serotype C adenoviruses. Consistent with this notion, the absenceor reduced expression of this receptor has been shown to correlate withthe poor sensitivity of certain cell types (e.g. lymphocytes, smoothmuscle cells) to adenovirus transduction (Leon et al, 1998, Proc. Natl.Acad. Sci. USA 95, 13159-13164; March et al., 1995, Hum. Gene Ther. 6,41-63). Moreover, numerous studies have now reported that primary tumorcells express only low levels of CAR (Li et al., 1999, Cancer Res. 59,325-330; Miller et al., 1998, Cancer Res, 58, 5738-5748).

The ability of adenoviruses to mediate infection of a broad spectrum ofdividing and non-dividing cell types constitutes an advantage overalternative gene transfer vectors. However, this broad tissue tropismmay also turn disadvantageous when genes encoding potentially harmfulproteins (e.g. cytokines, cytotoxic proteins, suicide gene products) areexpressed in surrounding normal tissues. Moreover, the overall in vitroefficiency of gene delivery might be reduced by a significant dilutionof the virus in the organism due to the transduction of non-targetcells. The development of adenovirus vectors with defined targeted entrypathways would therefore greatly improve the safety and efficacy of somecurrent gene therapy strategies. Thus, targeting adenoviral vectors mayimprove gene therapy procedures by either enhancing infectivity totransduction refractory cells (e.g. primary tumor cells) or restrictingthe viral tropism to specific tissue(s) of interest.

In this regard, increasing efforts have been made during the last yearsto redirect the adenovirus tropism from its natural receptors tospecific cell surface molecules. Since interactions of fiber and pentonbase with their corresponding cellular receptors represent keydeterminants of the viral tropism, retargeting the adenovirus may inprinciple be achieved by genetically, immunologically or chemicallyaltering the capsid proteins (see for example WO94/10323 and for areview Barnett et al., 2002, Biochemica et Biophysica Acta 1575, 1-14).Such modifications aim to abolish the interaction of the virus with itsnatural receptors and to provide new ligands recognizing moleculesspecifically expressed on the targeted cells.

The Ad5 fiber protein is a long trimeric protein that protrudes from thevirion surface. Each fiber monomer consists of three regions: the tailwhich associates with the penton base protein, the shaft, the length ofwhich varies among various serotypes and is characterized by a repeatingmotif of approximately 15 residues (Green et al., 1983, EMBO J. 2,1357-1365 Signas et al., 1985; J. Virol. 53, 672-678), and the knobwhich interacts with the cellular receptors (Henry et al., 1994, J.Virol. 68, 5239-5246). In Ad2, the C-terminal 40 aa residues in the knoband the last shaft repeat are required for Ad2 fiber trimerization (Hongand Engler, 1996. J. Virol. 70, 7071-7078; Novelli and Boulanger, 1991,Virol. 185, 365-376).

The crystal structure of the Ad5 fiber knob has been determined fromprotein expressed in bacteria. It is a trimer with a three-bladedpropeller and a surface depression. Each knob monomer is organized as aneight-stranded antiparallel beta-sheet structure with loops and tunasconnecting the beta-sheets (Xia et al., 1994, Structure 2, 1259-1270).Four of the beta-sheets (C, B, A and J) constitute the V-sheet: whichfaces towards the virion. The four other beta-sheets (G, H, I and D)form the R sheet and are presumed to face the cellular receptor. The Vsheet seems to play an important role in the trimerization of the fiberstructure, while the R sheet is thought to be involved in theinteraction with the receptor.

Recently, specific mutations which eliminate the interaction with CARwere identified, demonstrating that the CAR binding site of the fiberknob domain can be mutated without adversely affecting the quaternarystructure and overall conformation of the purified recombinant protein(WO98/44121, WO01/16344 and WO01/38361). For example, fiber proteinscarrying amino acid substitutions in the AB loop (involving Ser408 andPro409), in the DG loop (e.g. involving Tyr 477, Tyr 491, Ala 494 or Ala503) and in beta-strand F (e.g. involving Leu 485) or having deletion oftwo consecutive amino acid in the DG loop were shown to alter CARbinding (Bewley et al., 1999, Science 286, 1579-1583; Kirby et al.,1999, J. Virol 73, 9508-9514; Kirby et al., 2000, J. Virol. 74,2804-2813; Leissner et al., 2001, Gene Ther. 8, 49-57). Extending thesedata, it has been shown that viable and fully maturated viruses,carrying trimeric fibers mutated in the CAR binding domain, can begenerated (Leissner et al., 2001, Gene Ther. 8, 49-57; Roelvink et al.,1999, Science 286, 1568-1571; Jakubczak et al., 2001, J. Virol. 75,2972-2981). These viruses are structurally identical to native virusesand therefore constitute appropriate substrates for the insertion oftargeting ligands in the mutated fibers. In this respect, some specificlocations in the fiber protein have been identified for incorporation ofa novel targeting ligand into the fiber knob domain.

For example, addition of 24 amino acids containing the Gastrin ReleasingPeptide at the C-terminal end of the fiber did not prevent fibertrimerization (Michael et, al., 1995, Gene Ther. 2, 660-668). Similarly,addition at the same location of peptides of various lengths (17, 21 or32 amino acids) was shown to yield viable viruses (Wickham et al., 1997,J. Virol 71, 8221-8229). Several groups have reported that insertion ofstretches of lysine residues at the C-terminal end of the knob couldlead to the generation of high titer viruses that were characterized bya 10 to 300 fold increase in their efficiency of infection ofCAR-deficient cells, such as macrophages, endothelial cells, smoothmuscle cells or T lymphocytes (Wickham et al., 1997, J. Virol 71,8221-8229; Yoshida et al., 1998, Hum. Gene Ther. 9, 2503-2515; Wickhamet al., 1996, Nature Biotechnology 14, 1570-1573; Bouri et al., 1999,Hum. Gene Ther. 10, 1633-1640).

Apart from the carboxy-terminal end of the fiber, Krasnykh et al.demonstrated that the HI loop in the knob domain could be used tosuccessfully insert targeting ligands up to at least 63 amino acidswithout altering viral viability (Krasnykh et al., 1998, J. Virol. 72,1844-1852; Krasnykh et al., 2000, Cancer Res. 60, 6784-6787). Forinstance, insertion of a RGD motif in the HI loop was shown to expandthe tropism of the vector via the utilization of a CAR-independent cellentry mechanism (Dmitriev et al., 1998, J. Virol. 72, 9706-9713),allowing an enhancement in gene delivery to different primary tumors.Furthermore, the addition of this motif into the HI loop was shown toalter the transgene expression profile of systemically administeredvector, with a reduction of liver expression and simultaneous increasein the lung, heart and spleen (Reynolds et al., 1999, Gene Ther. 6,1336-1339). The introduction of a peptide ligand binding the transferrinreceptor in the HI loop facilitated gene transfer to cells whichover-express this receptor (Xia et al., 2000, J. Virol. 74,11359-11366). Similarly, a HUVEC cell-binding peptide allowed asignificant increase of the transduction efficiency of the retargetedvector towards these cells which are normally refractory to transduction(Nicklin et al., 2000, Circulation 102, 231-237).

Such mutated adenoviral vectors show reduced transduction ofCAR-expressing cells in vitro but retain significant CAR-independentinfectivity in vivo. It has been presumed that residual transductioncould be mediated through interaction between the adenoviral penton baseprotein and cellular integrins. More recently, doubly ablated adenoviralvectors, lacking both CAR and integrin binding capacities were proposedto abolish adenovirus native tropism (Einfeld et al., 2001, J. Virol.75, 11284-11291; Van Beusechem et al., 2001, J. Virol. 76, 2753-2762).

Thus, the prior art is deficient in mutated adenoviral fiber proteinsthat allow for reduction of the interaction with alternative cellularreceptors (other than CAR and integrins) which are involved inadenovirus attachment or internalization, and especially with the newlyidentified primary receptor for adenovirus, heparan sulfateglycosaminoglycans (HSG). The present invention fullfills thislong-standing need and desire in the art.

Therefore, the present invention provides novel mutants of theadenoviral fiber which allow, in particular, the production of viralparticles having the following properties:

-   -   (i) the adenoviral particles comprising said modified fiber lack        or substantially exhibit a substantially reduced binding to at        least sialic acid-containing receptors and/or        glycosaminoglycan-containing receptors, such as heparin/heparan        sulfate-containing receptors, and more particularly to HSG        receptors. The host specificity of these adenoviral particles        bearing the modified fiber is decreased or even inhibited, in        comparison to the host specificity of the adenoviral particles        carrying a nonmutated (i.e. wild-type) fiber.    -   (ii) When the mutated adenoviral particles also comprises        mutation(s) abolishing CAR binding, the adenoviral particles        comprising said doubly mutated fiber lack or exhibit a        substantially reduced binding to both the CAR receptor and the        sialic acid and/or glycosaminoglycan-containing receptors, such        as heparin/heparan sulfate-containing receptors, and more        particularly to HSG receptors. Altering interactions with both        CAR and HSG receptors may be essential to significantly restrict        the native tropism of an adenoviral. Such a particle represents        the best candidate for a basic vector that could be redirected        by incorporation of specific targeting ligands.    -   (iii) When the adenoviral particle comprising said modified        fiber also comprises a ligand specific for a cell-surface        anti-ligand (e.g. a tumor-specific or tissue-specific antigen),        it is possible to confer a novel tropism for one or more        specific cell types exhibiting at its (their) surface said        anti-ligand, in comparison to the nonmutated adenoviral        particles.

The present invention has, in particular, the advantage of providingnovel adenoviral particles, the properties of which make it possible todecrease the therapeutic amount of adenoviral particles to beadministered, to reduce dilution in the host organism and to target theviral infection to the cells to be treated. This host specificity isparticularly essential when an adenoviral vector expressing a cytotoxicgene is used, in order to avoid the propagation of the cytotoxic effectto healthy and nontargeted cells/tissues. In addition, the teachings ofthe present invention, allow other targeting systems intended fordeveloping methods of treatment relying on recombinant viral andnonviral vectors.

Other and further aspects, features and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

Accordingly, the present invention relates to a modified adenoviralfiber containing at least one mutation affecting one or more amino acidresidue(s) of said adenoviral fiber interacting with at least oneglycosaminoglycan and/or sialic acid-containing cellular receptor.

The term “and/or” whereever used herein includes the meaning of “and”,“or” and “all or any other combination of the elements connected by saidterm”.

The term “about” or “approximately” as used herein means within 20%,preferably within 10%, and more preferably within 5% of a given value orrange.

The term

amino acid

and residues are synonyms. This term refers to natural, unnatural and/orsynthetic amino acids, including D or L optical isomers, modified aminoacids and amino acid analogs.

The term

mutation

refers to a deletion, substitution or addition of one or more residues,or any combination of these possibilities. When several mutations arecontemplated, they can concern consecutive residues and/or nonconsecutive residues. Mutation can be made in a number of ways known tothose skilled in the art using recombinant techniques, includingenzymatically cutting from the fiber-encoding sequence followed bymodification and ligation of defined fragment, or by site-directedmutagenesis, especially by the Sculptor™ in vitro mutagenesis system(Amersham, Les Ullis, France) or by PCR techniques. Deletion mutationcan comprise from about 1 to 20 amino acid residues, preferably notexceeding 11 amino acids. Deletion of one to three amino acids arepreferred. According to a preferred embodiment, the mutation is asubstitution of at least one amino acid residue by another. It ispreferred that the mutation alters the charge of the substituted aminoacid residue.

The

adenoviral fiber

as used herein refers to the structural protein present at the surfaceof an adenoviral capsid (also called pIV), which is known to mediate theearly contact between virus and cells. The present invention encompassesthe full length adenoviral fiber which is encoded by the complete codingsequence (i.e. from the initiator ATG codon to the stop codon). However,it is possible to employ a fragment thereof generated by internaldeletion, or truncation having the properties as described herein. Forillustrative purpose, the fiber-encoding sequence can be isolated froman adenoviral genome by conventional recombinant techniques. The fibergene is present at the right end of the adenoviral genome positionedbetween E3 and E4 regions, e.g. from nucleotide (nt) 31042 to nt 32787in the Ad5 genome and from nt 31030 to nt 32778 in the Ad2 genome.

The modified adenoviral fiber of the invention may originate (beobtained) from an adenovirus of human or animal origin (e.g. canine,avian, bovine, murine, ovine, porcine, feline, simien and the like) orbe an hybrid comprising fragments of diverse origins. For instance, theadenovirus can be of subgroup A (e.g. serotypes 12, 18, 31), subgroup B(e.g. serotypes 3, 7, 11, 14, 16, 21, 34, 35, 50), subgroup C (e.g.serotypes 1, 2, 5, 6), subgroup D e.g. serotypes 8, 9, 10, 13, 15, 17,19, 20, 22-30, 32, 33, 36-39, 42-47, 51), subgroup E serotype 4),subgroup F (serotype 40, 41), or any other adenoviral serotype.Preferably, however, the modified fiber of the invention originates froman adenovirus of subgroup C, with a special preference for Ad2 or Ad5serotype.

The fiber of various human and animal adenoviruses are available ondatabases (e.g. GenBank) and literature publications. By way ofinformation, mention is made of the GenBank and literature referencesfor the fiber sequence of human serotype 2 (AAA92223), 3 (CAA26029). 5(M 18369), 31 (CAA54050), 41 (X17016), 50 and 51 (De Jong et al., 1999,J. Clinical Microbiology 37, 3940), bovine BAV-3 (AF030154; see alsoWO98/59063 and Reddy et al., 1998, J. Virol. 72, 1394-1402) and canineCAV-2 (Rasmussen et al., 1995, Gene 159,279-280).

The fiber of Ad2 includes 582 amino acids (aa), the sequence of whichbeing disclosed in Herissé et al. (1981, Nucleic Acid Res. 9, 4023-4042;incorporated into the present application by reference). The Ad5 fibersequence was determined by Cliroboczek and Jacrot (1987, Virology 161,549-554; incorporated by reference), and is 581 amino acids longincluding the initiator Met residue (as shown in SEQ ID NO: 1).

The crystal structure of the knob domain of the Ad5 fiber was determinedby Xia et al. (1994, Structure 2, 1259-1270; incorporated by reference).For the purposes of the invention, the terms <<beta sheet and

loop

is as defined in Xia et al (1994). These terms are conventional in thefield of protein biochemistry, and are defined in fundamental works (seefor example Stryer, Biochemistry, 2^(nd) edition, Chap 2, p 11-39, EdFreeman and Company, san Francisco). More specifically, each knobmonomer includes 8 antiparallel beta sheets referred to as A to D and Gto J, and 6 major loops of 8 to 55 residues. For example, loop ABconnects beta sheet A to beta sheet B. It is indicated that minor sheetsE and F are Considered to form part of loop DG connecting beta sheets Dand G. By way of indication, Table 1 gives the location of thesestructures in the amino acid sequence of the wild-type Ad5 fiber, asshown in SEQ ID NO: 1, the +1 representing the Met initiator residue.TABLE 1 beta sheet loop nomenclature Residues Nomenclature residues A400 to 403 AB 404 to 418 B 419 to 428 — — C 431 to 440 CD 441 to 453 D454 to 461 DG 462 to 514 G 515 to 521 GH 522 to 528 H 529 to 536 HI 537to 549 I 550 to 557 IJ 558 to 572 J 573 to 578

In order to simplify the presentation of the present application, onlythe positions relating to Ad5 are specifically given. However, it iswithin the scope of those skilled in the art to adapt the presentinvention to other adenovirus fibers.

The one or more mutation(s) contained in the modified adenoviral fiberof the invention can affect amino acid residue(s) located in the tail,shaft and/or knob domains, with a special preference for the knob. Themodified fiber of the invention preferably comprises a tail, a shaft anda knob. The various fiber region can be of the same serotype.Alternatively, it is also possible to use a

chimeric

fiber protein. For example, the tail and the shaft can be of oneserotype (e.g. of a subgroup C adenovirus such as Ad2 or Ad5) and theknob can be of another serotype (e.g. of a subgroup B adenovirus such asAd3 or Ad7).

Within the context of the present invention, the term

glycosaminoglycan-containing cellular receptor

encompasses any cell-surface molecule consisting of a core proteincontaining one or more covalently linked glycosaminoglycan side chains(e.g. linear side chains to form a long filament of glycosaminoglycan).The term

glycosaminoglycan

is conventional in the field of the art and can be defined as comprisingdisaccharide repeating units containing a derivative of an amino sugar,either glucosamine or galactosamine, with at least one of the sugars inthe repeating units having a negatively charged carboxylate or sulfategroup. Suitable glycosaminoglycan include without limitation chondritinsulfate, keratan sulfate, heparin, heparan sulfate, dermatan sulfate andhyaluronate as well as their various isoforms.

Preferably, the glycosaminoglycan-containing cellular receptor is aheparin- or heparan sulfate-containing cellular receptor. The structureof Heparin and heparan sulfate is for example illustrated in FIG. 18-15of Biochemistry (4^(th) edition, Lubert Stryer; ed Freeman and Compagny,New York) and can be defined as a copolymer of glucosamine andglucuronic or iduronic acid with various sulfatations and/or acetylationmodifications. Heparan sulfate is like heparin except that it has fewerN- and O-sulfate groups and more N-acetyl. Heparin- and heparansulfate-containing cellular receptors have been widely illustrated inthe literature, for example in Liu and Thorp (2002, Medical ResearchReviews 22(1), 1-25; incorporated into the present application byreference). They are involved in many biological processes (e.g. bloodcoagulation, would healing, embryonic development, viral infections,etc.). The structure and the saccharide side chains of the variousheparan sulfates-containing receptors encompassed by the presentinvention can vary according to their tissue distribution or theirbiological activities. Such heparan sulfate-containing receptors can beidentified by conventional techniques in the art, combining techniquesfrom virology, carbohydrate biochemistry, molecular biology and massspectrometry.

In one preferred embodiment, the heparin or heparan sulfate-containingreceptor encompassed by the present invention is the heparan sulfateglycosaminoglycan (HSG) receptors which normally interact with thewild-type adenoviral fiber, to mediate adenovirus attachment to a hostcell. The HSG receptor is as defined in Dechecchi et al. (2000, Virology268,382-390 and 2001, J. Virol. 75, 8772-8780).

Within the context of the present invention, the term

sialic acid-containing cellular receptor

encompasses any cell-surface molecule consisting of a core proteincontaining one or more polysaccharide side chains, such polysaccharideincluding sialic acid. Of course such receptors can exhibit complexpattern of glycosylation, containing apart sialic acid additional anddiverse carbohydrate residues. The term

sialic acid

(also designated N-acetyl neuraminate) is conventional in the field ofthe art and denotes a 9 carbone sugar with a carboxylate group, whichformula is for example given in FIG. 18-18 of Biochemistry (4^(th)edition, Lubert Stryer; ed Freeman and Compagny, New York)

Any native amino acid residue mediating or assisting in the interactionbetween the fiber and a native glycosaminoglycan-containing cellularreceptor (more particularly the HSG receptor) and/or a sialicacid-containing cellular receptor is suitable for mutation. For example,the native amino acid residue(s) to modify may be involved in aconformational change associated with receptor binding. Alternatively,the mutation may result in a charge modification, a modification of aparticular chemical group or a post-translational modification whichalter the binding to the cellular receptor. The modified fiber of thepresent invention can be mutated at any number of such native amino acidresidues, so long as it retains its ability to trimerize. The amino acidresidue to be mutated can be within any region of the fiber (e.g. shaftand/or knob), and as far as the knob is concerned, within a beta sheetor within a loop connecting two beta sheets. It is also possible toreplace one or more residue(s) of a fiber originating from a firstadenovirus, said residue(s) mediating directly or indirectly binding toa native glycosaminoglycan (e.g. HSG), or sialic acid-containingreceptor with equivalent residue(s) originating from a fiber of a secondadenovirus not capable of interacting with such glycosaminoglycan orsialic acid-containing receptors.

Native amino acid residue to be mutated can be selected by any method inthe art. For example, the sequences from different adenoviral serotypes(which are known in the art) can be compared to deduce conservedresidues likely to mediate binding to glycosaminoglycan or sialicacid-containing cellular receptors, and more particularly to HSGreceptors. Alternatively, or in combination, the sequence can be mappedon three dimensional representation of the protein to deduce thoseresidues which are most likely responsible for such a binding. Theseanalysis can be aided by resorting to any common algorithm or programfor deducing protein structural function interaction. Alternatively,random mutation can be introduced into a cloned adenoviral fiberexpression cassette (e.g. by site-directed mutagenesis, PCRamplification by varying the concentration of divalent cations in thePCR reaction, the error rate of the transcripts can be largelypredetermined as described in Weiss et al., 1997, J. Virol. 71,4385-4394 or Zhou et al., 1991, Nucleic Acid Res. 19, 6052). The mutatedsequence then can be subcloned back in the template vector, thusgenerating a library of fibers, some of which will harbor mutationswhich diminish binding to glycosaminoglycan (e.g. HSG) and/or sialicacid-containing cellular receptors.

According to a preferred embodiment, the modified adenoviral fiber ofthe present invention has an affinity for said glycosaminoglycan and/orsialic acid-containing cellular receptor of at least about one order ofmagnitude less than a wild-type adenoviral fiber. The decrease orabolition of binding to glycosaminoglycan or sialic acid-containingcellular receptors, and in particular to the HSG receptors, can beevaluated by measuring infectivity or cell attachment provided by themodified fiber of the invention or virus particles harboring such amodified fiber, using the technique in the art. Monitoring can beautoradiography (e.g. employing radioactive viruses or radiolabeledfiber proteins), immunochemistry, or by measuring plaque formation,cytotoxicity or by evaluating gene delivery (e.g. using a reportergene). For instance, suitable techniques include infection experimentsof suitable cells carried out in the presence and in the absence of acompetitor (i.e. heparin in the context of heparin/heparansulfate-containing receptors or sialic acid in the context of sialicacid-containing receptors as described in the Experimental Section ofthe present application). For example, an adenovirus deficient oraltered for HSG binding will be less or not competited by the competitoras compared to a wild-type adenovirus for infection of HSG-expressingcells. Indeed, after incubation of wild type adenovirus particles withheparin, the HSG-mediated pathway is inhibited due to the saturation ofthe wild type fiber with the competitor, whereas the infectivity ofparticles displaying a modified fiber of the invention is notsubstantially modified by the competitor. The alteration of the naturalspecificity can also be studied by evaluation of cell attachment usingradiolabeled viruses (for example labeled with ³H thymidine, asdescribed in Roelvin et al., 1996, J. Virol. 70, 76.14-7621) orradiolabeled fibers recombinantly produced. Alternatively, the affinityof the modified fiber of the invention can also be assayed for itsability to bind a substrate (e.g. heparin in the context ofheparin/heparan sulfate-containing receptors or sialic acid in thecontext of sialic acid-containing receptors) immobilized oil anappropriate support using the Biacore technique. It is also possible toevaluate infectivity after pretreatment of suitable cells by heparinase(in the context of heparin/heparan sulfate-containing receptors) orsialidase (in the context of sialic acid-containing receptors).

The ability of the modified fiber of the present invention to bind toglycosaminoglycan (e.g. HSG receptors) or sialic acid-containingcellular receptors is substantially decreased or abolished, when theresidual infection of cells containing such receptors with an adenovirusbearing such a modified fiber, is at least about one order of magnitudeless than that observed with an adenovirus bearing a wild-typeadenoviral fiber. Preferably, it is at least about two orders ofmagnitude, more preferably at least about three orders of magnitude,even more preferably at least about four orders of magnitude less thanthat observed with the corresponding wild-type adenovirus.

In one embodiment, the modified adenoviral fiber of the presentinvention is characterized in that it, comprises at least one mutationaffecting one or more residues within the shaft and/or the knob, andespecially within the AB loop, the CD loop, the DG loop and/or the betasheet I of the knob. Of course, the modified fiber of the invention cancombine several mutations which take place in one or more of theprecited regions, e.g. in the knob AB loop and/or the CD loop and/or theDG loop and/or the beta sheet I.

Advantageously, the amino acid residue(s) to be mutated in the modifiedfiber of the invention is (are) within about 5 amino acids of an aminoacid corresponding to residues 404-406, 449-454, 505-512, 551-560 of thewild-type Ad5 fiber (SEQ ID NO: 1). It is within the scope of thoseskilled in the art to identify the equivalent positions of these Ad5fiber residues in another adenoviral fiber, oil the basis of availablesequence database (see for example FIG. 9 of Xia et al., 1994, Structure2, 1259-1270 giving alignment of the fiber knob regions of Ad2, Ad5,Ad3, Ad7, Ad40, Ad41 and CAV or Van Raaij, 1999, Virology 262(2), 333).More preferably, the mutation affects one or more amino acid residue(s)selected from the group of residues consisting of the threonine inposition 404, the alanine in position 406, the valine in position 452,the lysine in position 506, the histidine in position 508, and theserine in position 555 of the wild type Ad5 fiber protein as shown inSEQ ID NO: 1. Even more preferably, the modified fiber protein of theinvention comprises at least one substitution mutation of a residuecorresponding to residues 404, 406, 452, 506, 508, and/or 555 of thewild-type Ad5 fiber (SEQ ID NO:1). Most preferably, said mutation of theAd5 fiber comprises:

-   -   the substitution of the threonine in position 404 by a small        aliphatic residue, such as alanine, proline or glycine, with a        special preference for glycine,    -   the substitution of the alamine in position 406 by a basic        residue such as lysine, arginine or histidine, with a special        preference for lysine,    -   the substitution of the valine in position 452 by a basic        residue such as lysine, arginine or histidine, with a special        preference for lysine,    -   the substitution of the lysine in position 506 by a slightly        basic amide residue such as glutamine or asparagine, with a        special preference for glutamine,    -   the substitution of the histidine in position 508 by a basic        residue such as lysine or arginine, with a special preference        for lysine, or    -   the substitution of the serine in position 555 by a basic        residue such as lysine, arginine or histidine, with a special        preference for lysine,    -   Or any combination thereof.

As mentioned before, the present invention also encompasses a modifiedadenoviral fiber having more than one mutation. It could be advantageousto mutate two or more residues involved in the interaction withglycosaminoglycan (e.g. HSG receptors) and/or sialic acid-containingreceptors, in order to further reduce or completely abolish its bindingcapability to one or both of these receptors. For example, the mutationof the glycine in position 450 by a lysine, of the threonine in position451 by an asparagine and of the valine in position 452 by a lysine isadvantageous in this respect. To further illustrate, mention can be madeof the following examples of a fiber of Ad5, comprising:

-   -   the substitution of the lysine in position 506 by glutamine and        the substitution of the histidine in position 508 by lysine        (K506Q/H508K);    -   the substitution of the threonine in position 404 by glycine,        the substitution of the lysine in position 506 by glutamine and        the substitution of the histidine in position 508 by lysine        (T404G/K506Q/H508K);    -   the substitution of the alanine in position 406 by lysine, the        substitution of the lysine in position 506 by glutamine and the        substitution of the histidine in position 508 by lysine        (A406K/K506Q/H508K);    -   the substitution of the valine in position 452 by lysine, the        substitution of the lysine in position 506 by glutamine and the        substitution of the histidine in position 508 by lysine        (V452K/K506Q/H508K);    -   the substitution of the lysine in position 506 by glutamine, the        substitution of the histidine in position 508 by lysine and the        substitution of the serine in position 555 by lysine        (K506Q/H508K/S555K);

Other combinations such as T404G/A406K, T404G/V452K, T404G/K506Q,T404G/H508K, T404G/S555K, A406K/V452K, A406K/K506Q, A406K/H508K,A406K/S555K, A452K/K506Q, A452K/H1508K; A452K/S555K, K506Q/S555K,H508K/S555K, T404G/A406K/V452K, T404G/A406K/K506Q, T404G/A406K/H508K,T404G/A406K/S555K, T404G/V452K/K506Q, T404G/V452K/H508K,T404G/V452K/S555K, T404G/K506Q/S555K,T404G/A406K/V452K/K506Q/H508K/S555K etc. are also contemplated, by thepresent invention.

As another alternative, the modified adenoviral fiber of the inventionoriginates (is obtained) from the wild type Ad2 fiber protein, andcomprises at least one mutation of one or more amino acid residue(s)selected from the group of residues consisting of the threonine inposition 404, the aspartic acid in position 406, the valine imposition452, the lysine in position 506, the glutamine in position 508, and thethreonine in position 556 of the wild type Ad2 fiber protein. Even morepreferably, the modified fiber protein of the invention comprises atleast one substitution mutation of a residue corresponding to residues404, 406, 452, 506, 508, or 556 of the wild-type Ad2 fiber. Suchsubstitutions of the Ad2 precited residues can be made by the type ofresidues as defined above for Ad5.

In accordance with the present invention, the modified adenoviral fiberof the present invention may further include at least one additionalmodification (e.g. amino acid substitution and/or deletion) other thanthose above-described. However, it is preferable not to drasticallymodify the three dimensional structure of the adenoviral fiber in orderto preserve its trimerization properties and its function in thematuration of the corresponding viral particles. In this context, theamino acids forming a special structure (e.g. a bend) will be replacedwith residues forming a similar structure, such as those mentioned inXia et al. (1994). This make it possible to maintain the structure ofthe modified fiber of the invention, while at the same time conferingupon it a property (e.g. host specificity) corresponding to that of thesecond adenovirus.

According to an advantageous embodiment, the modified adenoviral fiberof the present invention, further comprises at least one additionalmutation affecting one or more amino acid residue(s) interacting withthe CAR cellular receptor. In this regard and preferably, said modifiedadenoviral fiber has an affinity for said CAR cellular receptor and saidglycosaminoglycan (e.g. HSG receptor) and/or sialic acid-containingcellular receptor of at least about one order of magnitude less than awild-type adenoviral fiber, especially in the trimeric form. As before,the term

mutation

refers to deletion, addition or substitution or any combination thereof,with a special preference for substitution. Preferably the mutationaimed to abolish or reduce CAR binding affects one or more residue(s)located in the AB loop and/or the CD loop of the modified fiber of theinvention.

As indicated in Xia et al. (1994), the host specificity of Ad2 and Ad5is different from that of Ad3 and Ad7 with respect to CAR-mediatedpathway. Thus, it would be advantageous to replace one or moreresidue(s) of a subgroup C (e.g. Ad5 or Ad2) fiber involved inCAR-binding with one or more residue(s) located in an equivalentposition of a subgroup B (e.g. Ad3 or Ad7) fiber, so as to decrease theability of said fiber to bind the CAR receptor. By way of illustration,suitable CAR-ablating mutations include those described in WO98/44121,WO01/6344, WO/0138361 and WO00/15823 as well as in Kirby et al. (2000,J. Virol. 74, 2804-2813) and Leissner et al. (2001, Gene Ther. 8,49-57).

Preferably, the additional mutation (aimed to reduce or abolishCAR-binding) affects one or more residue(s) selected from the groupconsisting of the serine in position 408, the proline in position 409,the arginine in position 412, the lysine in position 417, the lysine inposition 420, the tyrosine in position 477, the arginine in position481, the leucine in position 485, the tyrosine in position 491, thealanine in position 494, the phenylalanine in position 497, themethionine in position 498, the proline in position 499 and the alaninein position 503 of the wild type Ad5 fiber protein (SEQ ID NO: 1). Evenmore preferably, the additional mutation is a substitution mutation ofone or more residue corresponding to residues 408, 409, 412, 417, 420,477, 481, 485, 491, 494, 497, 498, 499, or 503 of the wild type Ad5fiber protein (SEQ ID NO: 1), and most preferably the additionalmutation comprises:

-   -   the substitution of the serine in position 408 by glutamic acid        (S408E),    -   the substitution of the proline in position 409 by lysine        (P409K),    -   the substitution of the tyrosine in position 477 by alanine        (Y477A),    -   the substitution of the leucine in position 485 by lysine        (L485K),    -   the substitution of the tyrosine in position 491 by aspartic        acid (Y491D),    -   the substitution of the alanine in position 494 by aspartic acid        (A494D),    -   the substitution of the phenylalanine in position 497 by        aspartic acid (F497D),    -   the substitution of the methionine in position 498 by aspartic        acid (M498D),    -   the substitution of the proline in position 499 by glycine        (P499G),    -   the substitution of the alanine in position 503 by aspartic acid        (A503D), or    -   any combination thereof.

The modified adenoviral fiber of the invention can combine anymutation(s) affecting binding to native glycosaminoglycan (e.g. HGSreceptors) and/or sialic acid-containing receptors and any additionalmutation(s) affecting binding to CAR. Combination of the single S408E orA494D or A503D or the double A494D/A503D mutation affecting CAR bindingand of the double K506Q/H508K or the triple T404G/K506Q/H508K, orA406K/K506Q/H508K or V452K/K506Q/H508K or K506Q/H508K/S556K mutationaffecting HSG binding are suitable in the context of the presentinvention. Preferred examples include without limitation a modifiedadenoviral fiber comprising (i) the substitutiton of the serine inposition 408 by glutamic acid, the substitutiton of the lysine inposition 506 by glutamine and the substitutition of the histidine inposition 508 by lysine (S408E/K506Q/H508K), (ii) the substitutiton ofthe alanine in position 503 by aspartic acid, the substitutiton of thelysine in position 506 by glutamine and the substitutiton of thehistidine in position 508 by lysine (A503D/K506Q/H1508K), (iii) thesubstitutiton of the serine in position 408 by glutamic acid and thesubstitutiton of the serine in position 555 by lysine (S408E/S555K), or(iv) the substitutiton of the alanine in position 503 by aspartic acidand the substitutiton of the serine in position 555 by lysine(A503D/S555K).

The decrease or abolition of binding to CAR receptor provided by themodified fiber of the invention can be evaluated by infectivity or cellattachment as described above (e.g. cell attachment studies employingradiolabeled viruses or radiolabeled fibers recombinantly produced,Biacore techniques, immunochemistry, measurement of plaque formation,cytotoxicity, or gene delivery (e.g. using a reporter gene). It is alsopossible to probe a replica lift with radiolabeled CAR. Such techniquesare described for example in WO01/16344 and WO01/38361 and Leissner etal. (2001, Gene Ther. 8, 49-57). For instance, infectivity can bestudied in CAR+ cells (e.g. 293 cells or CHO cells transfected with aCAR-expressing plasmid) in the presence and in the absence of acompetitor (i.e. soluble knob or anti-knob antibody). Infectivity orcell attachment of an adenovirus deficient or altered for CAR bindingwill not be substantially modified in the presence or in the absence ofthe competitor, whereas infectivity or cell attachment of a non modified(e.g. wild-type) adenovirus will be dramatically decrease in thepresence of the competitor.

Advantageously, the ability of the modified fiber of the presentinvention to bind to the CAR is substantially decreased or abolished,when the residual infection of CAR+ cells measured with an adenovirusbearing such a modified fiber, is at least about one order of magnitudeless than that observed with the wild type adenovirus. Preferably, it isat least about two orders of magnitude, more preferably at least aboutthree orders of magnitude, even more preferably at least about fourorders of magnitude less than that observed with the wild typeadenovirus.

The modified adenoviral fiber of the invention can be further modifiedfor example in the shaft region.

Preferably, the modified adenoviral fiber of the invention trimerizeswhen produced in an eukaryotic host cell.

The modified adenoviral fiber protein of the invention can be producedby any suitable method. For example, the modified adenoviral fiber canbe synthetized using standard direct peptide synthesis techniques (e.g.as summarized in Bodanszky, 1984, Principle of Peptide Synthesis;Springer-Verlag, Heidelberg), such as via solid-phase synthesis (e.g.Merrifield, 1963, J. Am. Chem. Soc. 85, 2149-2154 and Barany et al.,1987, Int. J. Peptide Protein Res. 30, 705-739). Alternatively,oligonucleotide site-specific mutagenesis procedures are alsoappropriate to introduce the desired mutation(s) following cloning thesequence encoding a wild-type adenoviral fiber protein or peptidefragment into a vector (Bauer et al., 1985, Gene 37, 73 and Sculptor™ invitro mutagenesis system, Amersham, Les Ullis France). Alternatively,site-specific mutation(s) can be introduced by PCR techniques. Oneengineered, the sequence encoding the modified adenoviral fiber proteinor peptide fragment thereof can be subcloned into an appropriate vectorusing well known molecular techniques.

The present invention also relates to peptide fragment of the modifiedfiber protein of the invention. Within the context of the presentinvention, the term “peptide fragment” is intended to encompass peptidecomprising at least a minimum of 6 consecutive amino acids of themodified fiber protein, preferably at least about 10, more preferably atleast about 20, even more preferably at least about 40, and mostpreferably at least about 60, such consecutive amino acids bearing atleast one of the mutation described herein. When such a peptide fragmentis incorporated in place of an equivalent peptide fragment of a givenwild-type adenoviral fiber, it confers a reduced affinity for a nativeglycosaminoglycan (e.g. HSG) and/or sialic acid-containing cellularreceptor of at least about one order of magnitude less than said givenwild-type adenoviral fiber, in particular in trimeric form.

The present invention also relates to a trimer comprising the modifiedadenoviral protein as defined above.

Any suitable assay can be employed to evaluate its ability to trimerizeand/or associate with penton base. For example, the modified adenoviralfiber can be produced by standard recombinant techniques and theseproperties can be tested on the recombinant product. Any appropriatecloning or expression vector and corresponding suitable host cells canbe used in the context of the present invention, including but notlimited to bacteria (e.g. Escherichia coli), yeast, mammalian or insecthost cell systems and established cell lines.

One assay for trimerization is evaluation of its solubility since it wasshown that improperly folded monomers are generally insoluble (InProtein Purification, 3^(rd) Ed., 1994, Chap 9, p 270-282;Springer-Verlag, New york). Determination of the fiber solubility can beperformed on radiolabelled recombinant fiber protein, followingincorporation of radioactive amino acids into the protein duringsynthesis. Lysate from the host cell expressing the recombinant modifiedadenoviral fiber can be centrifuged and the supernatant and pellet canbe assayed via a scintillation counter. Trimerization can also beevaluated by Western blot analysis (e.g. on SDS-PAGE gel) carried out onthe supernatant and pellet obtained from cell lysate. Comparison of theamount of fiber protein detected from the pellet (insoluble) vis a visthe fiber protein detected from the supernatant (soluble) indicateswhether the modified adenoviral fiber is soluble. Alternatively,trimerization can be assayed by using a monoclonal antibody recognizingonly the trimeric form (e.g. via immunoprecipitation, Western blotting,etc.) (see for example Henry et al., 1994, J. Virol. 68, 5239-5246).Another evaluation of trimerization is the ability of the modified fiberto form a complex with the penton base (Novelli and Boulanger, 1995,Virol. 185, 1189), since only trimers can interact. This propensity canbe assayed by co-immunoprecipitation, gel mobility-shift assays,SDS-PAGE, etc. Another measurement is to detect the difference inmolecular weight of a trimer as opposed as a monomer. For example aboiled and denatured trimer will run as a lower molecular weight than anon-denatured stable trimer (Hong and Angler, 1996, J. Virol. 70,7071-7078).

According to a preferred embodiment, the trimer according to theinvention has an affinity for native glycosaminoglycan and/or sialicacid-containing receptors, and especially HSG receptors, of at leastabout one order of magnitude less than a wild type adenoviral fibertrimer. Methods for such measurement are indicated previously.Preferably, affinity for the trimer of the invention is at least abouttwo orders of magnitude, more preferably at least about three orders ofmagnitude, even more preferably at least about four orders of magnitudeless than that observed with the corresponding wild-type trimer.

According to another preferred embodiment, the trimer according to theinvention containing a modified adenoviral fiber having additionalmutation(s) as previously defined, further has an affinity for a nativeCAR cellular receptor of at least about one order of magnitude less thana wild type adenoviral fiber trifler. Reduction of CAR binding can beassayed as described above.

The present invention also relates to a DNA fragment or an expressionvector encoding the modified fiber of the invention or a fragmentthereof.

Within the context of the present invention, the term “DNA fragment” and“polynucleotide” are used interchangeably and define a polymeric form ofany length of deoxyribonucleotides (DNA). The DNA fragment of thepresent invention can be linear or circular. It may also comprisemodified nucleotides, such as methylated nucleotides or nucleotideanalogs (see U.S. Pat. No. 5,525,711, U.S. Pat. No. 4,711,955 or EPA 302175 as examples of modifications). If present, modifications to thenucleotide structure may be imparted before or after assembly of thepolymer (such as by conjugation with a labeling component). The sequenceof nucleotides may also be interrupted by non-nucleotide elements. TheDNA fragment of the present invention can code for a full lengthmodified fiber of an adenovirus serotype and also encompassesrestriction endonuclease-generated and PCR-generated fragments that canbe obtained therefrom. The present invention also encompasses syntheticfragments (e.g. produced by oligonucleotide synthesis).

Any type of vector can be used in the context of the present invention,whether of plasmid or viral, integrating or nonintegrating origin. Suchvectors are commercially available or described in the literature.Similarly, those skilled in the art are capable of adjusting theregulatory elements required for the expression of the DNA fragment ofthe invention. Preferably, said vector is an adenoviral vector capableof producing under suitable culturing conditions, adenoviral particlesbearing at their surface a modified fiber according to the presentinvention (as described hereinafter).

The present invention also relates to an adenoviral particle lacking awild-type fiber and comprising the modified adenoviral fiber proteinaccording to the present invention, and especially the trimer of theinvention. The modified adenoviral fiber protein can be expressed fromthe adenoviral genome itself or provided in trans by a complementationcell line, such as one defined hereinafter. The adenoviral particle hasa reduced capacity to interact with native glycosaminoglycan (e.g. HSG)and/or sialic acid-containing cellular receptors as compared to awild-type particle, due to the above-mentioned reduction in affinity ofthe fibers present in said particle.

Moreover, the adenoviral particle of the invention can be furthermodified to exhibit reduced affinity for native cellular receptor(s)other than glycosaminoglycan (e.g. HSG) or sialic acid containingreceptors, which are also involved in adenovirus attachment and/or entryinto the permissive cells. In this context, the adenoviral particle ofthe invention can be further modified through the inclusion ofadditional mutation(s) in the modified fiber or in other viralprotein(s) present at the surface of the particle. As discussed above,the adenoviral particle can include at least one additional mutationaffecting one or more amino acid residue within a region of theadenoviral fiber interacting with the CAR cellular receptor, to alsoreduce its ability to interact with the CAR cellular receptor. Also theadenoviral particle of the invention can further comprise one or morepenton base having a mutation affecting at least one native RGDsequence, preferably lacking a native RGD sequence, to reduce cellbinding or entry via cellular integrins (see e.g. U.S. Pat. Nos.5,559,099 and 5,731,190). But it has been observed that the integrinpathway is also inhibited with adenoviral particles exhibiting at theirsurface a trimer of modified fibers of the invention.

According to a preferred embodiment, the adenoviral particle of theinvention, further comprises a ligand, for example, for targetinginfection to a desired cell or cell population since the precitedmodification(s) alter(s) the native adenovirus tropism. Additionally,the ligand can be used to purify the virus, to inactivate the virus(e.g. by adsorbing it to a substrate for the ligand), or to grow thevirus on cell lines having the receptors recognized by said ligand.

For the purpose of the present invention, the term

ligand

defines any entity capable of recognizing and binding, preferably withhigh affinity, an anti-ligand. It is evident by reading thespecification that said ligand binds at least one cell-surfaceanti-ligand other than a native cellular receptor which normallymediates attachment and/or uptake of a wild type adenovirus (e.g. CAR,glycosaminoglycan (e.g. HSG) and/or sialic acid-containing cellularreceptors). This anti-ligand can be expressed or exposed at the surfaceof a particular cell, the targeting of which is desired. It may beadvantageous to target more particularly a tumor cell, an infected cell,a specific cell type or a category of cells. Therefore, suitableanti-ligands include without limitation polypeptides selected from thegroup consisting of cell-specific markers, tissue-specific receptors,cellular receptors, antigenic peptides (e.g. presented byhistocompatibility antigens), tumor-associated markers, tumor-specificreceptors, disease-specific antigens (e.g. viral antigens), and antigensspecifically expressed on the surface of the target cells, etc. Such ananti-ligand can be naturally exposed at the surface of the targeted cellor subsequent to a modification of said target cell (e.g. upon treatmentfor example to reduce glycosylation or phosphorylation). The anti-ligandlocalized at the surface of a target cell is preferably one that a wildtype adenoviral particle does not bind or binds but with a lowerspecificity than a adenoviral particle of the present invention. Thebinding specificity between a ligand and its corresponding anti-ligandcan be determined according to techniques of the art, including ELISA,immunofluorescence and surface plasmon resonance-based technology(Biacore AB).

According to the invention, the ligand is localized on the surface ofthe claimed adenoviral particle in general, the ligand that may be usedin the context of the present invention are widely described in theliterature; it is a moiety able to confer to the adenoviral particle ofthe invention, the ability to bind to a given anti-ligand or a class ofanti-ligands localized at the surface of at least one target cell.

In accordance with the aims pursued by the present invention, a ligandcan be a lipid, a glycolipid, an hormone, a sugar, a polymer (e.g. PEG,polylysine, PEI, etc.), a polypeptide, an oligonucleotide, a vitamin, anantigen, a lectin, a polypeptide moiety presenting targeting propertysuch as for example JTSI (WO94/40958), an antibody or combinationthereof. The term

antibody

include but are not limited to monoclonal antibodies, antibody fragments(such as for example Fab and dAb antibody fragments), single chainantibodies (scFv) and a minimal recognition unit thereof (i.e. afragment still presenting an antigenic specificity) such as thosedescribed in detail in immunology manuals (see for example Immunology,3rd edition 1993, Roitt, Brostoff and Male, ed Gambli, Mosby). Suitablemonoclonal antibodies to selected antigens may be prepared by knowntechniques, for example those disclosed in “Monoclonal Antibodies: Amanual of techniques”, H. Zola (CRC Press, 1988) and in “MonoclonalHybridoma Antibodies: Techniques and Applications”, J. G. R. Hurrell(CRC Press, 1982). Suitably prepared non-human antibodies may be“humanized” in known ways, for example by inserting the CDR regions ofmouse antibodies into the framework of human antibodies. Additionally,as the variable heavy (VH) and variable light (VL) domains of theantibody are involved in antigen recognition, variable domains of rodentorigin may be fused to constant domains of human origin such that theresultant antibody retains the antigenic specificity of the rodentparental antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81,6851-6855).

Alternatively, the ligand in use in the present invention can be derivedfrom various types of combinatorial libraries, using well knownstrategies for identifying ligands (see U.S. Pat. No. 5,622,699). Oneapproach uses recombinant bacteriophages to produce large libraries, asdescribed in Scott and Smith, 1990, Science 249, 386-390; Cwirla et al.,1990, Proc. Natl. Acad. Sci. USA 87, 6378-6382; Devlin et al., 1990,Science 249, 404-406). A second approach uses primarily chemicalmethods, such as the Geysen method (Geysen et al., 1986, MolecularImmunology 23, 709-715; Geysen et al., 1987, J. Immunologic Method 102,259-274) or the method of Fodor et al. (1991, Science 251, 767-773).Furka et al. (1991, Int. J. Peptide Protein res. 37, 487-493), U.S. Pat.No. 4,631,211 and U.S. Pat. No. 5,010,175 describe methods to produce amixture of peptides that can be tested as targeting ligands. In anotheraspect, synthetic libraries (Needels et al., 1993, Proc. Natl. Acad.Sci. USA 90, 10700-10704; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci.USA 90, 10922-10926; WO92/00252 and WO94/28028) can be used to screenfor targeting ligands.

Preferably, the ligand used in the present invention is a polypeptidehaving a minimal length of 6 amino acids. It is either a nativepolypeptide or a polypeptide derived from a native polypeptide.“Derived” means containing (i) one or more modifications with respect tothe native sequence (e.g. addition, deletion and/or substitution of oneor more residues), (ii) amino acid analogs, including not naturallyoccurring amino acids or (iii) substituted linkages as well as (vi)other modifications known in the art. The ligand can comprises sequencesof various origins (e.g. a peptide that selectively bind a cell-surfaceanti-ligand fused to a protease recognition site) or sequence which arenot contigous in the chain of amino acids in a given protein. In thiscontext, it could be advantageous to use a ligand mimicking theparticular conformation of a protein, e.g. in such a way to bringcontigous and noncontigous sequences in mutual proximity. Preferably,the ligand does not comprise an oligomerization domain in order to notinterfer with trimerization of the adenoviral fiber. In addition, theligand may have a linear or cyclized structure (e.g. by flanking at bothextremities a polypeptide ligand by cysteine residues). Additionally,the ligand moiety in use in the invention may include modifications ofits original structure by way of substitution or addition of chemicalmoieties (e.g. glycosylation, alkylation, acetylation, amidation,phosphorylation, addition of sulfhydryl groups and the like). Theinvention further contemplates modifications that render the liganddetectable. For this purpose, modifications with a detectable moiety canbe envisaged (i.e. a scintigraphic, radioactive, fluorescent, or dyelabels and the like). Suitable radioactive labels include but are notlimited to Tc^(99m), I¹²³ and In¹¹¹. Such detectable labels may beattached to the ligand by any conventional techniques and may be usedfor diagnostic purposes (e.g. imaging of tumoral cells).

In one embodiment, the ligand allows to target a virally infected celland is capable of recognizing and binding to a viral component (e.g.envelope glycoprotein, viral epitope) or capable of interfering with thevirus biology (e.g. entry, replication . . . ). For example, thetargeting of a HIV (Human Immunodeficiency Virus) infected cell can beperformed with a ligand specific for an epitope of the HIV envelope,such as a ligand consisting of or derived from the 2F5 antibody(Buchacher et al., 1992, Vaccines 92, 191-195) recognizing a highlyconserved epitope of the transmembrane glycoprotein gp41 or with aligand moiety interferring with HIV attachment to its cellular receptorCD4 (e.g. the extracellular domain of the CD4 molecule). Suitableligands also include those capable of recognizing and binding tocancer-associated viruses, such as human papilloma virus (HPV)associated with cervical cancer (e.g. by using a ligand directed to anHPV polypeptide including E6 and E7 early polypeptides as well as L1 andL2 late polypeptides), Epstein-Barr virus (EBV) associated withBurkitt's lymphomas (Evans et al., 1997, Gene Therapy 4, 264-267; e.g.by using a ligand directed to the EBV EBNA-1 antigen), polyoma virus,Hepatitis virus (e.g. by using a ligand directed to the E2 envelopepolypeptide of the hepatitis C virus, Chan et al., 1996, J. Gen. Virol.77, 2531). Such ligands are for example single chain antibodiesrecognizing one or more epitopes present in a viral envelope or core.

In another and preferred embodiment, the ligand allows to target atumoral cell and is capable of recognizing and binding to a moleculerelated to the tumoral status, such as a tumor-specific antigen, acellular protein differentially or over-expressed in tumoral cells or agene product of a cancer-associated virus (as described above).

Examples of tumor-specific antigens include but are not limited to MUC-1related to breast cancer (Hareuveni et al., 1990, Eur. J. Biochem 189,475-486), the products encoded by the mutated BRCA1 and BRCA2 genesrelated to breast and ovarian cancers (Miki et al., 1994, Science 226,66-71; Futreal et al., 1994, Science 226, 120-122; Wooster et al., 1995,Nature 378, 789-792), APC related to colon cancer (Polakis, 1995, Curr.Opin. Genet. Dev. 5, 66-71), prostate specific antigen (PSA) related toprostate cancer (Stamey et al., 1987, New England J. Med. 317, 909),carcinoma embryonic antigen (CEA) related to colon cancers (Schrewe etal., 1990, Mol. Cell. Biol. 10, 2738-2748), tyrosinase related tomelanomas (Vile et al., 1993, Cancer Res. 53, 3860-3864), receptor formelanocyte-stimulating hormone (MSH) which is expressed in high numberin melanoma cells, ErbB-2 related to breast and pancreas cancers (Harriset al., 1994, Gene Therapy 1, 170-175), and alpha-foetoprotein relatedto liver cancers (Kanai et al., 1997, Cancer Res. 57, 461-465). Forexample, a suitable ligand for targeting MUC-1 positive tumor cells canbe a fragment of an antibody capable of recognizing and binding to theMUC-1 antigen, such as the scFv fragment of the SM3 monoclonal antibodywhich recognizes the tandem repeat region of the MUC-1 antigen (Burshellet al., 1987, Cancer Res. 47, 5476-5482 Girling et al., 1989, Int J.Cancer 43, 1072-1076; Dokurno et al., 1998, J. Mol. Biol. 284, 713-728).

Examples of cellular proteins differentially or overexpressed in tumorcells include but are not limited to the receptor for interleukin 2(IL-2) overexpressed in some lymphoid tumors, for GRP (Gastrin ReleasePeptide) overexpressed in lung carcinoma cells, pancreas, prostate andstomach tumors (Michael et al., 1995, Gene Therapy 2, 660-668), TNF(Tumor Necrosis Factor) receptor, epidermal growth factor receptors, Fasreceptor, CD40 receptor, CD30 receptor, CD27 receptor, OX-40, alphavintegrins (Brooks et al., 1994, Science 264, 569) and receptors forcertain angiogenic growth factors (Hanahan, 1997, Science 277, 48).Based on these indications, it is within the scope of those skilled inthe art to define an appropriate ligand moiety capable of recognizingand binding to such proteins. To illustrate, IL-2 is a suitable ligandmoiety to bind to IL-2 receptor.

In still another preferred embodiment, the ligand in use in the presentinvention allows to target tissue-specific molecules. A particularanti-ligand can be present on a narrow class of cell types or a broadergroup encompassing several cell types. The adenoviral particle of theinvention can be targeted to cells within any organ or system, includingfor example, respiratory system (trachea, upper airways, lower airways,alveoly), nervous system and sensitory organs (e.g. skin, ear, nasal,tongue, eye), digestive system (e.g. oral epithelium, salivary glands,stomach, small intestines, duodenum, colon, gall bladder, pancreas,rectum), muscular system (e.g. cardiac muscle, skeletal muscle, smoothmuscle, connective tissue, tendons, etc), immune system (e.g. bonemarrow, stem cells, spleen, thymus, lymphatic system, etc), circulatorysystem (e.g. muscles connective tissue, endothelia of the arteries,veins, capillaries, etc), reproductive sytem (e.g. testis, prostate,cervix, ovaries), urinary system (e.g. bladder, kidney, urethra),endocrine or exocrine glands (e.g. breast, adrenal glands, pituitaryglands), etc.

For example, ligands suitable for targeting liver cells include but arenot limited to those derived from ApoB (apolipoprotein) able to bind tothe LDL receptor, alpha-2-macroglobulin able to bind to the LPRreceptor, alpha-1 acid glycoprotein able to bind to theasialoglycoprotein receptor and transferrin able to bind to thetransferrin receptor. A ligand moiety for targeting activatedendothelial cells may be derived from the sialyl-Lewis-X antigen (ableto bind to ELAM-1), from VLA-4 (able to bind to the VCAM-1 receptor) orfrom LFA-1 (able to bind to the ICAM-1 receptor). A ligand derived fromCD34 is useful to target the hematopoietic progenitor cells throughbinding to the CD34 receptor. A ligand derived from ICAM-1 is moreintended to target lymphocytes through binding to the LFA-1 receptor.The targeting of T-helper cells may use a ligand derived from HIV gp-120or a class II MHC antigen capable of binding to the CD4 receptor. Thetargeting of neuronal, glial, or endothelial cells can be performedthrough the use of ligands directed for example to tissue factorreceptor (e.g. FLT-1, CD31, CD36, Cd34, CD105, CD13, ICAM-1; McCormicket al., 1998, J. Biol. Chem. 273, 26323-26329), thrombomodulin receptor(Lupus et al., 1998, Suppl. 2 S120, VEGFR-3 (Lymboussaki et al., 1998,Am. J. Pathol. 153, 395-403), VCAM-1 (Schwarzacher et al., 1996,Artherosclerosis 122, 59-67) or other receptors. The targeting of bloodclots can be made via fibrinogen or aIIbb3 peptide. Finally, inflamedtissues can be targeted through selectins, VCAM-1, ICAM-1, etc.

Moreover, suitable ligands also include linear stretches of amino acids,such as polylysine, polyarginine and the like recognized by integrins.Also, a ligand can comprise a commonly employed tag peptide (e.g. shortamino acids sequences known to be recognized by available antisera),such as sequences from glutathione-S-transferase (GST) from Shistosomamanosi, thioredoxin beta galactosidase, or maltose binding protein (MPB)from E. coli, human alkaline phosphatase, the FLAG octapeptide,hemagluttinin (HA).

It will be appreciated by those skilled in the art that ligand moietieswhich are polypeptides may be conveniently made using recombinant DNAtechniques. The ligand moiety may be fused to a protein on the surfaceof the adenoviral particle of the invention or may be synthesizedindependently (e.g. by de novo synthesis or by expression of theencoding sequence in an eukaryotic or prokaryotic cell) and then coupledto the adenoviral particle as disclosed below. The nucleic acidsequences encoding many of the ligands encompassed by the presentinvention are known, for example those for peptide hormones, growthfactors, cytokines and the like and may readily be found by reference topublically accessible nucleotide sequence databases such as EMBL andGenBank. Many cDNAs encoding peptide hormones, growth factors, all orpart of antibodies, cytokines and the like, all of which may be usefulas ligands, are generally commercially available. Once the nucleotidesequence is known it is obvious to the person skilled in the art how tomake the sequence encoding the chosen ligand using, for example,chemical DNA synthetic techniques or by using the polymerase chainreaction to amplify the required DNA from genomic DNA or fromtissue-specific cDNA.

Once a suitable ligand is identified, it can be incorporated into anylocation of the adenoviral particle of the invention, provided that itis still capable of interacting with its respective anti-ligand. In thecontext of the invention, said ligand is immunologically, chemically orgenetically coupled to a viral polypeptide exposed at the surface ofsaid adenoviral particle. Said viral polypeptide exposed at the surfaceof the adenoviral particle is selected from the group consisting ofpenton base, hexon, fiber, protein IX, protein VI and protein IIIa.

Chemical coupling of the selected ligand to the surface of theadenoviral particle may be performed directly through reactivefunctional groups (e.g. thiol or amine groups) or indirectly by a spacergroup or other activating moiety. In particular, coupling may be donewith (i) homobifunctional or (ii) heterobifunctional cross-linkingreagents, with (iii) carbodiimides, (iv) by reductive amination or (vi)by activation of carboxylates (see for example Bioconjugate techniques1996; ed G Hermanson; Academic Press).

Homobifunctional cross linkers including glutaraldehyde andbis-imidoester like DMS (dimethyl suberimidate) may be used to coupleamine groups of the ligand to lipid structures containing diacyl amines.Many heterobifunctional cross linkers may be used in the presentinvention, in particular those having both amine reactive andsulfhydryl-reactive groups, carbonyl-reactive and sulfhydryl-reactivegroups and sulfhydryl-reactive groups and photoreactive linkers.Suitable heterobifunctional crosslinkers are described in Bioconjugatetechniques (1996) 229-285; ed. G Hermanson; Academic Press) andWO99/40214. Examples of the first category include but are not limitedto SPDP (N-succinimidyl 3-(2-pyridyidithio)propionate), SMBP(succinimidyl-4-(p-maleimidophenyl)butyrate), SMPT(succinimidyloxycarbonyl-alpha-methyl-(alpha-2-pyridyidithio)toluene),MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB(N-succinimidyl (4 iodoacetyl) aminobenzoate), GMBS(gamma-maleimidobutyryloxy) succinimide ester), SIAX(succinimidyl-6-iodoacetyl amino hexonate, SIAC(succinimidyl-4-iodoacetyl amino methyl), NPIA (p-nitrophenyliodoacetate). The second category is useful to couplecarbohydrate-containing molecules (e.g. env glycoproteins, antibodies)to sulfydryl-reactive groups. Examples include MPBH (4-(4-Nmaleimidophenyl)butyric acid hydrazide) and PDPH(4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide (M₂C₂H and3-2(2-pyridyldithio)proprionyl hydrazide). As an example of the thirdcategory, one may cite ASIB (1-(pazidosalicylamido)-4-(iodoacetamido)butyrate). Another alternativeincludes the thiol reactive reagents described in Frisch et al.(Bioconjugate Chem. 7 (1996) 180-186).

Chemical coupling between the ligand and the adenoviral particle of theinvention may also be performed using a polymer such as polyethyleneglycol (PEG) or its derivatives (see for example WO99/40214;Bioconjugate Techniques, 1996, 606-618; ed G Hermanson; Academic Pressand Frisch et al., 1996, Bioconjugate Chem. 7, 180-186). The chemicalcoupling may also be non covalent, for example via electrostaticinteractions (e.g. between a cationic ligand and a negatively chargedadenoviral particle) or through the use of affinity components such asProtein A, biotin/avidin, which are able to associate both partners.

Immunological coupling involves antibodies to conjugate the selectedligand to the adenoviral particle of the invention For example, it ispossible to use biotinylated antibodies directed to a surface-exposedviral epitope and streptavidin-labelled antibodies directed against theselected peptide ligand according to the technique disclosed by Roux etal. (1989, Proc. Natl. Acad Sci USA 86, 9079). Bifunctional antibodiesdirected against each of the coupling partners are also suitable forthis purpose.

According to a preferred embodiment, the selected ligand is geneticallycoupled to the is adenoviral particle of the invention. Advantageously,the sequence encoding said ligand is inserted in the adenoviral genome,preferably within a gene encoding an adenoviral polypeptide localized atthe surface. The present invention also encompass the use of specificsignals (e.g. a membrane anchoring polypeptide) and peptide spacer (orlinker) to further improve presentation of the ligand at the surface ofthe adenoviral particle. The term

peptide spacer

or

linker

as used herein refers to a peptide sequence of about one to 20 aminoacids that is included to connect the ligand to the adenoviralpolypeptide. The spacer is preferably made up of amino acid residueswith high degrees of freedom of rotation, which permits the ligand toadopt a conformation that is recognized by its anti-ligand partner.Preferred amino acids for the spacer are alanine, glycine, prolineand/or serine. In specific embodiments, the spacer is a peptide havingthe sequence Ser-Ala, Pro-Ser-Ala or Pro-Gly-Ser or a repetitionthereof.

According to a first alternative, a portion of the surface-exposedadenoviral polypeptide can be removed and the ligand is inserted inreplacement of the deleted portion. According to a second alternative,the ligand-encoding sequence is inserted in the viral sequence encodingthe surface-exposed adenoviral polypeptide. Ligand insertion can be madeat any location, at the N-terminus, the C-terminus or between two aminoacid residues of the viral polypeptide. Preferably the insertion is madein frame and does not disrupt the viral open reading frame.

More preferably, the ligand is genetically coupled to a viralpolypeptide exposed at the surface of the adenoviral particle of theinvention, selected from the group consisting of penton base, hexon,fiber, protein IX, protein VI and protein IIIa at any suitable location.Where the ligand is inserted or replace a portion of the penton base,preferably it is within the hypervariable regions to ensure contact withthe anti-ligand. Where the ligand is inserted or replace a portion ofthe hexon, preferably it is within the hypervariable regions. A suitableexample is an adenovirus hexon comprising a deletion of about 13 aminoacid residues from the HVR5 loop, corresponding to about amino acidresidue 269 to about amino acid residue 281 of the Ad5 hexon andinsertion of the ligand at the site of the deletion, eventuallyconnected by a first spacer at the N-terminus and a second spacer at theC-terminus of the ligand. Even more preferably, the ligand isgenetically inserted in the modified fiber of the invention, especiallyat the C-terminus or within the HI loop. More specifically, insertion inthe HI loop may be made between about amino acid residue 538 to aboutamino acid residue 548 of the Ad5 fiber and insertion of the ligand atthe site of the deletion, eventually connected by a first spacer at theN-terminus and a second spacer at the C-terminus of the ligand.Insertion at the C-terminus of the adenoviral fiber is generally madejust upstream of the stop codon, optionally through the use of a peptidespacer connected at the N-terminus of the ligand. In a general manner,the insertion site is selected in such a way to maximally presentationof the ligand to the anti-ligand and to not disturb the interactionbetween the other viral proteins and fiber trimerization. Also, theligand can be genetically inserted in the pIX protein, at any mocationbut with a special preference for insertion at the C-terminus or withinthe C-terminal portion of pIX (e.g. in replacement of or in addition toone or more residues located within the 40 pIX residues preceeding theSTOP codon). Where the ligand is inserted in the pIX protein, preferablypIX is also mutated in the coil coiled domain (as described for examplein Rosa-Calavatra et al., 2001, J. Virol. 75, 7131-7141).

Of course, the adenoviral particle of the present invention can comprisemore than one ligand, each binding to a different anti-ligand. Forexample, an adenoviral particle can comprise a first ligand permittingaffinity-based purification and a second ligand that selectively bind acell surface anti-ligand as described herein.

According to a particular case of the invention, the adenoviral particleof the invention is an

empty

capsid, i.e. it contains no nucleic acid. The use of such empty capsidis illustrated for example, for implementing DNA-based gene therapyprotocols. In this respect, WO95/21259 describes a method forintroducing a nucleic acid into a cell, using a combination ofadenoviral particles and nucleic acids (e.g. naked nucleic acids). Thismethod is based mainly on the capacity of the adenoviral particles totransport molecules to the cell nucleus after endocytosis. Curiel et al.(1992, Hum. Gene Ther. 3, 147-154) and Wagner et al; (1992, Proc. Natl.Acad. Sci. USA 89, 6099-6103) showed that complexation of plasmids withinactivated adenoviral particles allows the endosomes to be lysed beforefusion with the lysosomes, therefore allowing the plasmids to escapedegradation. This results in a 100- to 1000-fold increase intransfection efficiency in vitro.

According to a preferred embodiment, the adenoviral particle of thepresent invention comprises an adenoviral genome (reference will be alsomade to an adenoviral virus or adenoviral particle or adenovirus).

In one embodiment, the adenoviral genome is engineered to beconditionally replicative (CRAd adenovirus), in order to replicateselectively in specific cells (e.g. proliferative cells) as describedfor example in Heise and Kim (2000, J. Clin. Invest. 105, 847-851).

In another and preferred embodiment, the adenoviral genome isreplication-defective, i.e. incapable of autonomous replication in theabsence of complementation. The deficiency is obtained by a mutation ordeletion of one or more viral gene(s) essential to the replication. Itis preferably defective for at least the E1 function by total or partialdeletion and/or mutation of one or more genes constituting the E1region. Advantageously, the E1 deletion covers nucleotides (nt) 458 to3328 or 458 to 3510 by reference to the sequence of the human adenovirustype 5 disclosed in the Genebank database under the accession number M73260. Furthermore, the adenoviral backbone of the vector may compriseadditional modifications (deletions, insertions or mutations in one ormore other viral genes). An example of an E2 modification is illustratedby the thermosensible mutation affecting DBP (DNA Binding Protein)(Ensiniger et al., 1972, J. Virol. 10, 328-339). The adenoviral sequencemay also be deleted of all or part of the E4 region. A partial deletionretaining the ORFs 3 and 4 or ORFs 3 and 6/7 may be advantageous (seefor example European application EP 974 668; Christ et al., 2000, HumanGene Ther. 11, 415-427; Lusky et al., 1999, J. Virol. 73, 8308-8319).Additional deletions within the non-essential E3 region may increase thecloning capacity, however it may be advantageous to retain all or partof the E3 sequences coding for the polypeptides (e.g. gp19k) allowing toescape the host immune system (Gooding et al., 1990, Critical Review ofImmunology 10, 53-71) or inflammatory reactions (EP 1203819). Secondgeneration vectors retaining the ITRs and packaging sequences andcontaining substantial genetic modifications aimed to abolish theresidual synthesis of the viral antigens may also be envisaged, in orderto improve long-term expression of the expressed gene in the transducedcells (WO94/28152; Lusky et al., 1998, J. Virol 72, 2022-2032).

Adenoviruses adaptable for use in accordance with the present invention,can be derived from any human or animal source, in particular canine(e.g. CAV-1 or CAV-2; Genbank ref CAVIGENOM and CAV77082 respectively),avian (Genbank ref AAVEDSDNA), bovine (such as BAV3; Seshidhar Reddy etal., 1998, J. Virol. 72, 1394-1402), murine (Genbank ref ADRMUSMAV1),ovine, feline, porcine or simian adenovirus or alternatively from ahybrid thereof. Any serotype can be employed. However, the humanadenoviruses of the C sub-group are preferred and especiallyadenoviruses 2 (Ad2) and 5 (Ad5). Generally speaking, the cited virusesare available in collections such as ATCC and have been the subject ofnumerous publications describing their sequence, organization andbiology, allowing the artisan to apply them. It is preferred that theadenovirus be of the same subgroup or serotype that the adenovirus fromwhich originates the modified fiber protein of the invention.

According to another preferred embodiment, the adenoviral particle ofthe invention is recombinant, i.e. the adenoviral genome comprises atleast one gene of interest placed under the control of the regulatoryelements allowing its expression in a host cell.

The term “gene of interest” refers to a nucleic acid which can be of anyorigin and isolated from a genomic DNA, a cDNA, or any DNA encoding aRNA, such as a genomic RNA, an mRNA, an antisense RNA, a ribosomal RNA,a ribozyme or a transfer RNA. The gene of interest can also be anoligonucleotide (i.e. a nucleic acid having a short size of less than100 bp).

In a preferred embodiment, the gene of interest in use in the presentinvention, is a therapeutic gene, i.e. encodes a gene product oftherapeutic interest. A “gene product of therapeutic interest” is onewhich has a therapeutic or protective activity when administeredappropriately to a patient, especially a patient suffering from adisease or illness condition or who should be protected against adisease or condition. Such a therapeutic or protective activity can becorrelated to a beneficial effect on the course of a symptom of saiddisease or said condition. It is within the reach of the man skilled inthe art to select a gene encoding an appropriate gene product oftherapeutic interest, depending on the disease or condition to betreated. In a general manner, his choice may be based on the resultspreviously obtained, so that he can reasonably expect, without undueexperimentation, i.e. other than practicing the invention as claimed, toobtain such therapeutic properties.

In the context of the invention, the gene of interest can be homologousor heterologous with respect to to the host cell or organism into whichit is introduced. Advantageously, it encodes a polypeptide, a ribozymeor an antisense RNA. The term

polypeptide

is to be understood as any translational product of a polynucleotidewhatever its size is, and includes polypeptides having as few as 7 aminoacid residues (peptides), but more typically proteins. In addition, itmay be of any origin (prokaryotes, lower or higher eukaryotes, plant,virus etc). It may be a native polypeptide, a variant, a chimericpolypeptide having no counterpart in nature or fragments thereof.Advantageously, the genie of interest in use in the present inventionencodes at least one polypeptide that can compensate for one or moredefective or deficient cellular proteins in an animal or a humanorganism, or that acts through toxic effects to limit or remove harmfulcells from the body. A suitable polypeptide may also be immunityconferring and acts as an antigen, e.g. to provoke a humoral response.

Representative examples of polypeptides encoded by the gene of interestinclude without limitation polypeptides selected from the groupconsisting of:

-   -   polypeptides involved in the cellular cycle, such as p21, p16,        the expression product of the retinoblastoma (Rb) gene, kinase        inhibitors (preferably of the cycdin-dependent type), GAX,        GAS-1, GAS-3, GAS-6, Gadd45 and cyclin A, B and D;    -   angiogenic polypeptides, such as members of the family of        vascular endothelial growth factors (VEGF; i.e. heparin-binding        VEGF Genbank accession number M32977), transforming growth        factor (TGF, and especially TGFalpha and beta), epithelial        growth factors (EGF), fibroblast growth factor (FGF and        especially FGF alpha and beta), tumor necrosis factors (TNF,        especially TNF alpha and beta), CCN (including CTGF, Cyr61, Nov.        Elm-1, Cop-1 and Wisp-3), scatter factor/hepatocyte growth        factor (SH/HGF), angiogenin, angiopoietin (especially 1 and 2),        angiotensin-2, plasminogen activator (tPA) and urokinase (uPA);    -   cytokines, including interleukins (in particular IL-2, IL-6,        IL-8, IL-12), colony stimulating factors (such as GM-CSF, G-CSF,        M-CSF), interferons (such as IFN beta; Genbank accession number        M25460; IFN gamma; Genbank accession number M29383 or IFN        alpha);    -   chemokines, including RANTES, MIP alpha, MIP-1 beta, DCCK1, MDC,        IL-10 (Genbank accession number U 16720) and MCP-1;    -   polypeptides capable of decreasing or inhibiting a cellular        proliferation, including antibodies, toxins, immunotoxins,        polypeptides inhibiting an oncogen expression products (e.g.        ras, map kinase, tyrosine kinase receptors, growth factors), Fas        ligand (Genbank accession number U08137), polypeptides        activating the host immune system;    -   polypeptides capable of inhibiting a bacterial, parasitic or        viral infection or its development, such as antigenic        determinants, transdominant variants inhibiting the action of a        viral native protein by competition (EP 614980, WO95/16780),        immunoadhesin (Capon et al., 1989, Nature 337, 525-531; Bym et        al., 1990, Nature 344, 667-670), immunotoxins (Kurachi et al.,        1985, Biochemistry 24, 5494-5499) and antibodies (Buchacher et        al., 1992, Vaccines 92, 191-195);    -   enzymes, such as urease, renin, thrombin, metalloproteinase,        nitric oxide synthases (eNOS (Genbank accession number M95296)        and iNOS), SOD, catalase, heme oxygenase (Genbank accession        number X06985), enase, the lipoprotein lipase family; oxygen        radical scavengers;    -   enzyme inhibitors, such as alpha1-antitrypsin, antithrombin III,        plasminogen activator inhibitor PAI-1, tissue inhibitor of        metalloproteinase 1-4;    -   polypeptides capable of restoring at least partially a deficient        cellular function responsible for a pathological condition, such        as dystrophin or minidystrophin (in the context of myopathies;        England et al., 1990, Nature 343, 180-182), insulin (in the        context of diabetes), hemophilic factors (for the treatment of        hemophilias and blood disorders such as Factor VIIa (U.S. Pat.        No. 4,784,950), Factor VIII (U.S. Pat. No. 4,965,199) or        derivative thereof (U.S. Pat. No. 4,868,112 having the B domain        deleted) and Factor IX (U.S. Pat. No. 4,994,371)), CFTR (in the        context of cystic fibrosis; Riordan et al., 1989, Science 245,        1066-1072), erythropoïetin (anemia), lysosomal storage enzymes,        including glucocerebrosidase (Gaucher's disease; U.S. Pat. No.        5,879,680 and U.S. Pat. No. 5,236,838), alpha-galactosidase        (Fabry disease; U.S. Pat. No. 5,401,650), acid alpha-glucosidase        (Pompe's disease; WO00/12740), alpha n-acetylgalactosaminidase        (Schindler disease; U.S. Pat. No. 5,382,524), acid        sphingomyelinase (Niemann-Pick disease; U.S. Pat. No. 5,686,240)        and alpha-iduronidase (WO93/10244);    -   angiogenesis inhibitors, such as angiostatin, endostatin,        platelet factor-4;    -   transcription factors, such as nuclear receptors comprising a        DNA binding domain, a ligand binding domain and domain        activating or inhibiting transcription (e.g. fusion products        derived from oestrogen, steroid and progesterone receptors);    -   reporter genes (such as CAT, luciferase, eGFP . . . );    -   an antibody (whole immunoglobulins of any class, chimeric        antibodies and hybrid antibodies with dual or multiple antigen        or epitope specificities and fragments thereof such as F(ab)2,        Fab′, Fab, scFv including hybrid fragments and anti-idiotypes)        and    -   any polypeptides that are recognized in the art as being useful        for the treatment or prevention of a clinical condition.

It is within the scope of the present invention that the gene ofinterest may include addition(s), deletion(s) and/or modification(s) ofone or more nucleotide(s) with respect to the native sequence.

When the adenoviral particle of the present invention comprises a ligandaimed to target a tumor cell, the gene of interst preferably encodes ananti-tumor agent. A variety of anti-tumor agents may be utilized inaccordance with the present invention. Within the context of the presentinvention, “anti-tumor agents” are understood to refer to compounds ormolecules which inhibit the growth of a selected tumor. Representativeexamples of anti-tumor agents include immune activators and tumorproliferation inhibitors. Briefly, immune activators function byimproving immune recognition of tumor-specific antigens (e.g. throughhumoral and/or cellular-mediated immunity). As a result, the immunesystem will more effectively inhibit or kill tumor cells. Immuneactivation may be subcategorized into immune modulators (molecules whichaffect the interaction between lymphocyte and tumor cell) andlymphokines, that act to proliferate, activate, or differentiate immuneeffector cells. Representative examples of immune modulators includeCD3, ICAM-1, ICAM-2, LFA-1, LFA-3, beta-2-microglobulin, chaperones,alpha interferon and gamma interferon, B7/BB1 and majorhistocompatibility complex (MHC). Representative examples of lymphokinesinclude gamma interferon, tumor necrosis factor, IL-1, IL-2, IL-3, IL4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, GM-CSF, CSF-1, and G-CSF.

Tumor proliferation inhibitors act by directly inhibiting cell growth,or by directly killing the tumor cell. Representative examples of tumorproliferation inhibitors include toxins and suicide genes.Representative examples of toxins include without limitation ricin (Lambet al., 1985, Eur. J. Biochem. 148, 265-270), diphtheria toxin (Twetenet al., 1985, J. Biol. Chem. 260, 10392-10394), cholera toxin (Mekalanoset al., 1983, Nature 306, 551-557 Sanchez and Holmgren, 1989, Proc.Natl. Acad. Sci. USA 86, 481-485), gelonin (Stirpe et al., 1980, J.Biol. Chem. 255, 6947-6953), pokeweed (Irvin, 1983, Pharmac. Ther. 21,371-387), antiviral protein (Barbieri et al., 1982, Biochem. J. 203,55-59; Irvin et al., 1980, Arch. Biochem. Biophys. 200, 418-425; Irvin,1975, Arch. Biochem Biophys. 169, 522-528), tritin, Shigella toxin(Calderwood et al., 1987, Proc. Natl. Acad. Sci. USA 84, 4364-4368;Jackson et al., 1987, Microb. Path. 2, 147-153) and Pseudomonas exotoxinA (Carroll and Collier, 1987, J. Biol. Chem. 262, 8707-8711).

Suicide genes

can be defined in the context of the present invention as any geneencoding an expression product able to transform an inactive substance(prodrug) into a cytotoxic substance, thereby giving rise to cell death.The gene encoding the TK HSV-1 constitutes the prototype of the suicidegene family (Caruso et al., 1993, Proc. Natl. Acad. Sci. USA 90,7024-7028; Culver et al., 1992, Science 256, 1550-1552). While the TKpolypeptide is non toxic as such, it catalyzes the transformation ofnucleoside analogs (prodrug) such as acyclovir or ganciclovir. Thetransformed nucleosides are incorporated into the DNA chains which arein the process of elongation, cause interruption of said elongation andtherefore inhibition of cell division. A large number of suicidegene/prodrug combinations are currently available. Those which may morespecifically be mentioned are rat cytochrome p450 andcyclophosphophamide (Wei et al., 1994, Human Gene Ther. 5, 969-978),Escherichia coli (E. coli) purine nucleoside phosphorylase and6-methylpurine deoxyribonucleoside (Sorscher et al., 1994, Gene Therapy1, 223-238), E. coli guanine phosphoribosyl transferase and6-thioxanthine (Mzoz et al., 1993, Human Gene Ther. 4, 589-595).However, in a preferred embodiment, the adenoviral particle of theinvention comprises a suicide gene encoding a polypeptide having acytosine deaminase (CDase) or a uracil phosphoribosyl transferase(UPRTase) activity or both CDase and UPRTase activities, which can beused with the prodrug 5-fluorocytosine (5-FC). The use of a combinationof suicide genes, e.g. encoding polypeptides having CDase and UPRTaseactivities, can also be envisaged in the context of the invention.

CDase and UPRTase activities have been demonstrated in prokaryotes andlower eukaryotes, but are not present in mammals. CDase is normallyinvolved in the pyrimidine metabolic pathway by which exogenous cytosineis transformed into uracil by means of a hydrolytic deamination, whereasUPRTase transforms uracile in UMP. However, CDase also deaminates ananalog of cytosine, 5-FC, thereby forming 5-fluorouracil (5-FU), whichis highly cytotoxic when it is converted into 5-fluoro-UMP (5-FUMP) byUPRTase action.

Suitable CDase encoding genes include but are not limited to theSaccharomyces cerevisiae FCY1 gene (Erbs et al., 1997, Curr. Genet. 31,1-6; WO93/01281) and the E. coli codA gene (EP 402 108). SuitableUPRTase encoding genes include but are not limited to those from E. coli(upp gene; Anderson et al., 1992, Eur. J. Biochem. 204, 51-56),Lactococcus lactis (Martinussen and Hammer, 1994, J. Bacteriol. 176,6457-6463), Mycobacterium bovis (Kim et al. 1997, Biochem Mol. Biol. Int41, 1117-1124), Bacillus subtilis (Martinussen et al. 1995, J.Bacteriol. 177, 271-274) and Saccharomyces cerevisiae FUR-1 gene; Kernet al., 1990, Gene 88, 149-157). Preferably, the CDase encoding gene isderived from the FCY1 gene and the UPRTase encoding gene is derived fromthe FUR-1 gene.

The present invention also encompasses the use of mutant suicide genes,modified by addition, deletion and/or substitution of one or severalnucleotides providing that the cytotoxic activity of the gene product bepreserved. A certain number of CDase and UPRTase mutants have beenreported in the literature including a fusion protein which encodes atwo domain enzyme possessing both CDase and UPRTase activitiesWO96/16183) as well as a mutant of the UPRTase encoded by the FUR-1 genehaving the first 35 residues deleted (mutant FCU-1 disclosed inWO99/54481).

Additional examples of tumor proliferation inhibitors include antisensesequences which inhibit tumor cell growth by preventing the cellularsynthesis of critical proteins needed for cell growth. Examples of suchantisense sequences include antisense to positively-acting growthregulatory genes, such as oncogenes and protooncogenes (c-myc, c-fos,c-jun, c-myb, c-ras, Kc, JE, HER2), as well as antisense sequences whichblock any of the enzymes in the nucleotide biosynthetic pathway.Finally, tumor proliferation inhibitors also include tumor suppressorssuch as p53, retinoblastoma (Rb), and MCC and APC for colorectalcarcinoma.

Sequences which encode the above-described anti-tumor agents may beobtained from a variety of sources. For example, plasmids that containsequences which encode anti-tumor agents may be obtained from adepository such as the American Type Culture Collection (ATCC,Rockville, Md.), or from commercial sources such as BritishBio-Technology Limited (Cowley, Oxford England). Alternatively, knowncDNA sequences which encode anti-tumor agents may be obtained from cellswhich express or contain the sequences. Briefly, mRNA from a cell whichexpresses the gene of interest is reverse transcribed with reversetranscriptase using oligo dT or random primers. The single stranded cDNAmay then be amplified by PCR utilizing oligonucleotide primerscomplementary to sequences on either side of desired sequences.

As mentioned above, the gene of interest is operably linked toregulatory elements allowing its expression in the host cell (e.g. thecell to be treated). Such regulatory elements include a promoter thatmay be obtained from any viral, bacterial or eukaryotic gene (even fromthe gene of interest) and be constitutive or regulable. Optionally, itcan be modified in order to improve its transcriptional activity, deletenegative sequences, modify its regulation, introduce appropriaterestriction sites etc. Suitable promoters include but are not limited tothe followings: adenoviral E1a, MLP, PGK, MT (metallothioneine; Mc Ivoret al., 1987, Mol. Cell Biol. 7, 838-848), alpha-1 antitrypsin, CFTR,surfactant, immunoglobulin, beta-actin, SRalpha, SV40, RSV LTR,TK-HSV-1, SM22, Desmin (WO 96/26284) and early CMV.

Preferably, the regulatory elements allowing the expression of the geneof interest are functional within a host cell presenting at its surfacean anti-ligand to which the ligand in use in the invention binds. Saidregulatory elements comprise a promoter preferably selected from thegroup consisiting of tissue-specific promoters and tumor-specificpromoters. Suitable promoters include those functional in proliferativecells, such as those isolated from genes overexpressed in tumoral cells,such as the MUC-1 gene overexpressed in breast and prostate cancers(Chen et al., 1995, J. Clin. Invest. 96, 2775-2782), the CEA (CarcinomaEmbryonic Antigen)-encoding gene overexpressed in colon cancers (Schreweet al., 1990, Mol. Cell. Biol. 10, 2738-2748), the ERB-2 encoding geneoverexpressed in breast and pancreas cancers (Harris et al., 1994, GeneTherapy 1, 170-175) and the alpha-foetoprotein-encoding geneoverexpressed in liver cancers (Kanai et al., 1997, Cancer Res. 57,461-465).

Those skilled in the art will appreciate that the regulatory elementscontrolling the expression of the gene of interest may further compriseadditional elements for proper initiation, regulation and/or terminationof transcription and translation of the gene(s) of interest into thehost cell or organism. Such additional elements include but are notlimited to non coding exon/intron sequences, transport sequences,secretion signal sequences, nuclear localization signal sequences, IRES,polyA transcription termination sequences, tripartite leader sequences,sequences involved in replication or integration. Said elements havebeen reported in the literature and can be readily obtained by thoseskilled in the art. Illustrative examples of introns suitable in thecontext of the invention include those isolated from the genes encodingalpha or beta globin (i.e. the second intron of the rabbit beta globingene Green et al., 1988, Nucleic Acids Res. 16, 369; Karasuyama et al.,1988, Eur. J. Immunol. 18, 97-104), ovalbumin, apolipoprotein,immunoglobulin, factor IX, factor VII and CFTR and synthetic intronssuch as the intron present in the pCI vector (Promega Corp, pCImammalian expression vector E1731) made of the human beta globin donorfused to the mouse immunoglobin acceptor or the intron 16S/19S of SV40(Okayma and Berg, 1983, Mol. Cell. Biol. 3, 280-289). The additionalelements may also contain a polyadenylation signal operably linked tothe gene(s) of interest, to allow proper termination of thetranscription. It is preferably positioned downstream of the gene ofinterest.

The gene of interest in use in the present invention can be inserted inany location of the adenoviral genome, with the exception of thecis-acting sequences (ITRs and packaging sequences). Preferably, it isinserted in replacement of a deleted region (E1, E3 and/or E4), with aspecial preference for the deleted E1 region. In addition, theexpression cassette may be positioned in sense or antisense orientationrelative to the transcriptional direction of the region in question.

The present invention encompasses the use of one or more gene(s) ofinterest. In this regard, the combination of genes encoding a suicidegene product and a cytokine (such as IL-2, IL-8, IFNgamma, GM-CSF) maybe advantageous in the context of the invention. The different genes ofinterest may be controlled by common (polycistronic cassette) orindependent regulatory sequences that are positioned either in the sameor in opposite directions.

In addition, adenoviral particles or empty capsids of the invention canalso be used to transfer nucleic acids (e.g. a plasmidic vector) by aviris-mediated cointernalization process as described in U.S. Pat. No.5,928,944. This process can be accomplished in the presence of (a)cationic agent(s) such as polycarbenes or lipid vesicles comprising oneor more lipid layers.

The adenoviral particle of the invention may be prepared and propagatedaccording to any conventional technique in the field of the art (e.g. asdescribed in Graham and Prevect, 1991, Methods in Molecular Biology, Vol7, Gene Transfer and Expression Protocols; Ed E. J. Murray, The HumanPress Inc, Clinton, N.J. or in WO96/17070)

The invention also relates to a process for producing the adenoviralparticle according to the invention, comprising the steps of:

-   -   Introducing said adenoviral particle or the genome of said        adenoviral particle into a suitable cell line,    -   Culturing said cell line under suitable conditions so as to        allow the production of said adenoviral particle, and    -   Recovering the produced adenoviral particle from the culture of        said cell line, and    -   Optionally purifying said recovered adenoviral particles.

The adenoviral particle or its genome is introduced into the cell inaccordance with known techniques, such as transformation, transduction,microinjection of minute amounts of DNA into the nucleus of a cell(Capechi et al., 1980, Cell 22, 479-488), transfection for example withCaPO₄ (Chen and Okayama, 1987, Mol. Cell Biol. 1, 2745-2752),electroporation (Chu et al., 1987, Nucleic Acid Res. 15, 1311-1326),lipofection/liposome fusion (Felgner et al., 1987, Proc. Natl. Acad.Sci. USA 84, 7413-7417), particle bombardement (Yang et al., 1990, Proc.Natl. Acad. Sci. USA 87, 9568-9572), gene guns, infection (e.g. with aninfective viral particle), direct DNA injection (Acsadi et al., 1991,Nature 352, 815-818), microprojectile bombardment (Williams et al.,1991, Proc. Natl. Acad. Sci. USA 88, 2726-2730), or the like.

With respect to cell line, both prokaryotic and eukaryotic cells may beemployed, which include bacteria yeast, plants and animals, includinghuman cells. Preferably, the adenoviral particle isreplication-defective and said appropriate cell line complements atleast one defective function of said adenoviral particle, eventually incombination with a helper virus. The cell lines 293 (Graham et al.,1977, J. Gen. Virol. 36, 59-72) and PERC6 (Fallaux et al., 1998, HumanGene Therapy 9, 1909-1917) are commonly used to complement the E1function. Other cell lines have been engineered to complement doublydefective vectors (Yeh et al., 1996, J. Virol. 70, 559-565; Krougliakand Graham, 1995, Human Gene Ther. 6, 1575-1586; Wang et al., 1995, GeneTher. 2, 775-783; Lusky et al., 1998, J. Virol. 72, 2022-2033; EP919627and WO97/04119).

The present invention also encompasses a process for producingadenoviral particles lacking a functional fiber (by deleting all or partof the fiber-encoding sequence). In this case, the process of theinvention employs preferably a cell line expressing a modifiedadenoviral fiber of the invention. Such a cell line comprises either ina form integrated into the genome or in episome form a DNA fragment oran expression vector of the present invention. Of course, the DNAfragment is placed under the control of appropriate translational and/ortranscriptional regulatory elements to allow production of the modifiedadenoviral fiber of the invention in said cell line. Preferably, thiscell line is further capable of complementing an one or more adenoviralfunctions selected from the group consisting of the functions encoded bythe E1, E2, E4, L1, L2, L3, L4, L5 regions or any combination thereof.It is preferably produced from the 293 cell line or from the PER C6 cellline, e.g. by transfecting an expression vector encoding the modifiedfiber protein of the invention.

Alternatively, the process of the invention employs an adenoviral vectorof the invention which genome contains the sequence encoding a modifiedfiber of the invention in replacement of the native fiber gene and twocell lines. First the adenoviral vector is introduced in a first cellline providing appropriate complementation according to the viralbackbone (e.g. E1 for E1-deleted vectors) and further providing awild-type fiber (e.g. 293 or PER-C6 transfected with an expressionvector encoding the sequence encoding the corresponding wild-typefiber). This amplification step allows the recovery of adenoviralparticles having a genome comprising the modified fiber-encodingsequence packaged in capsid having a wild-type fiber. The resultingadenovirus particles recovered from the culture of said first cell lineare then used to infect a second cell line providing only the necessarycomplementation (e.g. 293 or PERC-6). Thus, after one round ofamplification in such a second cell line, the produced adenoviralparticles will be packaged in neosynthetized capsids comprising themodified fiber expressed from the adenoviral genome.

The adenoviral particles can be recovered from the culture supernatantbut also from the cells after lysis and optionally further purifiedaccording to standard techniques (e.g. chromatography,ultracentrifugation, as described in WO96/27677, WO98/00524 WO98/26048and WO00/50573).

The present invention also provides an eukaryotic host cell comprisingthe DNA fragment or the adenoviral particle of the present invention.

For the purpose of the invention, the term “host cells” should beunderstood broadly without any limitation concerning particularorganization in tissue, organ, etc or isolated cells of a mammalian(preferably a human). Such cells may be unique type of cells or a groupof different types of cells and encompass cultured cell lines, primarycells and proliferative cells from mammalian origin, with a specialpreference for human origin. Suitable host cells include but are notlimited to hematopoietic cells (totipotent, stem cells, leukocytes,lymphocytes, monocytes, macrophages, APC, dendritic cells, non-humancells and the like), pulmonary cells, tracheal cells, hepatic cells,epithelial cells, endothelial cells, muscle cells (e.g. skeletal muscle,cardiac muscle or smooth muscle), fibroblasts.

Moreover, according to a specific embodiment, the eukaryotic host cellof the invention can be further encapsulated. Cell encapsulationtechnology has been previously described (Tresco et al., 1992, ASAIO J.38, 17-23; Aebischer et al., 1996, Human Gene Ther. 7, 851-860).According to said specific embodiment, transfected or infected hostcells are encapsulated with compounds which form a microporous membraneand said encapsulated cells can further be implanted in vivo. Capsulescontaining the cells of interest may be prepared employing a hollowmicroporous membrane from poly-ether sulfone (PES) (Akzo Nobel Faser AG,Wuppertal, Germany; Deglon et al. 1996, Human Gene Ther. 7, 2135-2146).This membrane has a molecular weight cutoff greater than 1 MDa whichpermits the free passage of proteins and nutrients between the capsuleinterior and exterior, while preventing the contact of transplantedcells with host cells.

The present invention also relates to a composition comprising the hostcell or the adenovirus particle of the invention, or which is producedusing the process according to the invention, preferably apharmaceutical composition, in combination with a vehicle which isacceptable from a pharmaceutical point of view. In a special case, thecomposition may comprise two or more adenoviral particles or eukaryotichost cells, which may differ by the nature (i) of the regulatorysequence and/or (ii) of the gene of interest and/or (iii) of theadenoviral backbone and/or (iv) the ligand.

The composition according to the invention may be manufactured in aconventional manner for a variety of modes of administration includingsystemic, topical and localized administration (e.g. topical, aerosol,instillation, oral). For systemic administration, injection ispreferred, e.g. subcutaneous, intradermal, intramuscular, intravenous,intraperitoneal, intrathecal, intracardiac (such as transendocardial andpericardial), intratumoral, intravaginal, intrapulmonary, intranasal,intratracheal, intravascular, intraarterial, intracoronary orintracerebroventricular. Intramuscular, intravenous and intratumoralconstitute the preferred modes of administration. The administration maytake place in a single dose or a dose repeated one or several timesafter a certain time interval. The appropriate administration route anddosage may vary in accordance with various parameters, as for example,the condition or disease to be treated, the stage to which it hasprogressed, the need for prevention or therapy and/or the therapeuticgene to be transferred. As an indication, a composition based onadenoviral particles may be formulated in the form of doses of between10⁴ and 10¹⁴ iu (infectious units), advantageously between 10⁵ and 10¹³iu and preferably between 10⁶ and 10¹² iu. The titer may be determinedby conventional techniques. The composition of the invention can be invarious forms, e.g. in solid (e.g. powder, lyophilized form), liquid(e.g. aqueous).

Moreover, the composition of the present invention can further comprisea pharmaceutically acceptable carrier for delivering said adenoviralparticle or eukaryotic host cell into a human or animal body. Thecarrier is preferably a pharmaceutically suitable injectable carrier ordiluent which is non-toxic to a human or animal organism, at the dosageand concentration employed-(for examples, see Remington's PharmaceuticalSciences, 16^(th) ed. 1980, Mack Publishing Co). It is preferablyisotonic, hypotonic or weakly hypertonic and has a relatively low ionicstrength, such as provided by a sucrose solution. Furthermore, it maycontain any relevant solvents, aqueous or partly aqueous liquid carrierscomprising sterile, pyrogen-free water, dispersion media, coatings, andequivalents, or diluents (e.g. Tris-HCl, acetate, phosphate),emulsifiers, solubilizers or adjuvants. The pH of the pharmaceuticalpreparation is suitably adjusted and buffered in order to be appropriatefor use in humans or animals. Representative examples of carriers ordiluents for an injectable, composition include water, isotonic salinesolutions which are preferably buffered at a physiological or slightlybasic pH (between about pH 8 to about pH 9, with a special preferencefor pH8.5). Suitable buffer include phosphate buffered saline, Trisbuffered saline, mannitol, dextrose, glycerol containing or notpolypeptides or proteins such as human serum albumin). A particularlypreferred composition comprises an adenoviral particle in 1 Msaccharose, 150 mM NaCl 1 mM MgCl₂, 54 mg/l Tween 80, 10 mM Tris pH 8.5.Another preferred composition is formulated in 10 mg/ml mannitol, 1mg/ml HSA, 20 mM Tris, pH 7.2, and 150 mM NaCl. These compositions arestable at −70° C. for at least six months.

In addition, the composition according to the present invention mayinclude one or more

stabilizing

additive(s), capable of preserving its degradation within the human oranimal and/or of improving uptake into the host cell. Such additives maybe used alone or in combination and include hyaluronidase (which isthought to destabilize the extra cellular matrix of the host cells asdescribed in WO98/53853), chloroquine, protic compounds such aspropylene glycol, polyethylene glycol, glycerol, ethanol, 1-methylL-2-pyrrolidone or derivatives thereof, aprotic compounds such asdimethylsulfoxide (DMSO), diethylsulfoxide, di-n-propylsulfoxide,dimethylsulfone, sulfolane, dimethyl-formamide, dimethylacetamide,tetramethylurea, acetonitrile (see EP 890 362), cytokines, especiallyinterleukin-10 (IL-10) (PCT/EP/99 03082), nuclease inhibitors such asactin G (WO99/56784) and cationic salts such as magnesium (Mg²⁺) (EP998945) and lithium (Li⁺) (EP 99 40 3310.8) and any of theirderivatives. The amount of cationic salt in the composition of theinvention preferably ranges from about 0.1 mM to about 100 mM, and stillmore preferably from about 0.1 mM to about 10 mM. One may also employsubstances susceptible to facilitate gene transfer in arterial cells,such as a gel complex of poly-lysine and lactose (Midoux et al., 1993,Nucleic Acid Res. 21, 871-878) or poloxamer 407 (Pastore, 1994,Circulation 90, 1-517).

The composition of the present invention is particularly intended forthe preventive or curative treatment of disorders, conditions ordiseases associated with cancer. The term “cancer” encompasses anycancerous conditions including diffuse or localized tumors, metastasis,cancerous polyps and preneoplastic lesions (e.g. dysplasies) as well asdiseases which result from unwanted cell proliferation. A variety oftumors may be selected for treatment in accordance with the methodsdescribed herein. In general, solid tumors are preferred, althoughleukemias and lymphomas may also be treated especially if they havedeveloped a solid mass, or if suitable tumor associated markers existsuch that the tumor cells can be physically separated from nonpathogenicnormal cells. For example, acute lymphocytic leukemia cells, may besorted from other lymphocytes with the leukemia specific marker “CALLA”.Cancers which are contemplated in the context of the invention includewithout limitation glioblastoma, sarcoma, melanomas, mastocytoma,carcinomas (e.g. colorectal and renal cell carcinomas) as well asbreast, prostate, testicular, ovarian, cervix (in particular, thoseinduced by a papilloma virus), lung (e.g. lung carcinomas includinglarge cell, small cell, squamous and adeno-carcinomas), kidney, bladder,liver, colon, rectum, pancreas, stomac, esophagus, larynx, brain,throat, skin, central nervous system, blood (lymphomas; leukemia, etc.),bone, etc cancers.

The composition of the invention may also be used for the prevention andtreatment of other diseases, such as those affecting muscles, bloodvessels (preferably arteries) and/or the cardiovascular system,including without limitation ischemic diseases (peripheral, lower limb,cardiac ischemia and angina pectoris), artherosclerosis, hypertension,atherogenesis, intimal hyperplasia, (re)restenosis following angioplastyor stent placement, neoplastic diseases (e.g. tumors and tumormetastasis), benign tumors, connective tissue disorders (e.g. rheumatoidarthritis), ocular angiogenic diseases (e.g. diabetic retinopathy,macular degeneration, corneal graft rejection, neovascular glaucoma),cardiovascular diseases (myocardial infarcts), cerebral vasculardiseases, diabetes-associated diseases, immune disorders (e.g. chronicinflammation or autoimmunity), neurodegenerative diseases, Parkinsondiseases and genetic diseases (muscular dystrophies such as Becker andDuchenne, hemophilias, Gaucher's disease, cystic fibrosis, etc. aslisted above). Another application is to use the composition of theinvention as in vivo expression system for disorders that involve thegene product to be secreted into the bloodstream, especially to restoreprotein deficiencies (e.g. hemophilia by expressing the appropriatecoagulation factor, lysosomal storage diseases, anemias).

Moreover, in the composition of the invention, the adenoviral particleor the expression vector of the present invention may be conjugated to alipid or polymer. In this respect, preferred lipids or polymers arecationic to interact with cell membranes (Felgner et al., 1989, Nature337, 387-388). Cationic lipids or mixtures of cationic lipids which maybe used in the present invention include Lipofectin™, DOTMA:N-[1-(2,3-dioleyloxyl)propyl]-N,N,N-trimethylammonium (Feigner, 1987,Proc. Natl. Acad. Sci. USA 84, 7413-7417), DOGS:dioctadecylamidoglycylspemine or Transfectam™ (Behr, 1989, Proc. Natl.Acad. Sci. USA 86, 6982-6986), DMRIE:1,2-dimiristyloxypropyl-3-dimethyl-hydroxyethylammonium and DORIE:1,2-diooleyloxypropyl-3-dimethyl-hydroxyethylammnoium (Felgner, 1993,Methods 5, 67-75), DC-CHOL: 3[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (Gao, 1991, BBRC179, 280-285), DOTAP (McLachlan, 1995, Gene Therapy 2, 674-622),Lipofectamine™, spermine- and spermidine-cholesterol, Lipofectace™ (fora review see for example Legendre, 1996, Medecine/Science 12, 1334-1341or Gao, 1995, Gene Therapy 2, 710-722) and the cationic lipids disclosedin patent applications WO 98/34910, WO 98/14439, WO 97/19675, WO97/37966 and their isomers. Nevertheless, this list is not exhaustiveand other cationic lipids well known in the art can be used inconnection with the present invention as well. Cationic polymers ormixtures of cationic polymers which may be used in the present inventioninclude chitosan (WO98/17693), poly(aminoacids) such as polylysine (U.S.Pat. No. 5,595,897 or FR 2 719 316); polyquaternary compounds;protamine; polyimines; polyethylene imine or polypropylene imine (WO96/02655); polyvinylamines; polycationic polymer derivatized with DEAE,such as DEAE dextran (Lopata et al., 1984, Nucleic Acid Res. 12,5707-5717); polyvinylpyridine; polymethacrylates; polyacrylates;polyoxethanes; polythiodiethylaminomethylethylene (P(TDAE));polyhistidine; polyornithine; poly-p-aminostyrene; polyoxethanes;co-polymethacrylates (eg copolymer of HPMA;N-(2-hydroxypropyl)-methacrylamide); the compound disclosed in U.S. Pat.No. 3,910,862, polyvinylpyrrolid complexes of DEAE with methacrylate,dextran, acrylamide, polyimines, albumin, onedimethylaminomethylmethacrylates and polyvinylpyrrolidone-methylacrylaminopropyltrimethyl ammonium chlorides;polyamidoaminne (Haensler and Szoka, 1993, Bioconjugate Chem. 4,372-379); telomeric compounds (patent application filing number EP98401471.2); dendritic polymers (WO 95/24221). Nevertheless, this listis not exhaustive and other cationic polymers well known in the art canbe used in the composition according to the invention as well. Colipidsmay be optionally included in order to facilitate entry of the vectorinto the cell. Such colipids can be neutral or zwitterionic lipids.Representative examples include phosphatidylethanolamine (PE),phospliatidylcholine, phosphocholine, dioleylphosphatidylethanolamine(DOPE), sphingomyelin, ceramide or cerebroside and any of theirderivatives. The ratio of cationic lipids and/or cationic polymers tocolipid(s) (on a weight to weight basis), when the co-lipid(s) is (are)co-existing in the complex, can range from 1:0 to 1:10. In preferredembodiments, this ratio ranges from 1:0.5 to 1:4.

The complexation of the adenoviral particle or expression vector of theinvention with one or more of the above-cited compounds can be performedaccording to standard techniques. For example, the compound(s) (e.g.cationic lipids) is (are) dissolved in an appropriate organic solventsuch as chloroform. The mixture is then dried under vaccum. The filmobtained is further dissolved in an appropriate amount of solvent ormixture of solvents which are miscible in water, in particular ethanol,dimethylsulfoxide (DMSO), or preferably a 1:1 (v:v) ethanol:DMSOmixture, so as to form lipid aggregates according to a known method (WO96/03977), or alternatively, is suspended in an appropriate quantity ofa solution of detergent such as an octylglucoside (e.g.n-octyl-beta-D-glucopyranoside or6-O—(N-heptylcarbamoyl)-methyl-alpha-D-glucopyranoside). The suspensionmay then be mixed with a solution comprising the desired amount ofadenoviral particles. Subsequent dialysis may be carried out in order toremove the detergent and to recover the composition of the invention.The principle of such a method is described by Hofland et al. (1996,Proc. Natl. Acad. Sci. USA 93, 7305-7309).

The present invention also relates to the use of the expression vector,of the adenoviral particle or of the composition of the invention, or ofan adenoviral particle which is produced using the process according tothe invention, for the preparation of a drug intended for the treatmentor the prevention of a disease in a human or animal organism by genetherapy.

Within the scope of the present invention, “gene therapy” has to beunderstood as a method for introducing any expressible sequence into acell. Thus, it also includes immunotherapy that relates to theintroduction of a potentially antigenic epitope into a cell to induce animmune response which can be cellular or humoral or both.

In a preferred embodiment, such a use is suitable for the treatment orthe prevention of any of the diseases cited above, and more particularlycancer diseases. For this purpose, the adenoviral particle of thepresent invention may be delivered in vivo to the human or animalorganism by specific delivery means adapted to this pathology. In thiscontext, it is possible to operate via direct intratumoral injection.Alternatively, one may employ eukaryotic host cells that have beenengineered ex vivo to contain the adenoviral particle according to theinvention. Methods for introducing such elements into an eukaryotic cellare well known to those skilled in the art. The transfected/infectedcells are grown in vitro and then reintroduced into the patient. Thegraft of encapsulated host cells is also possible in the context of thepresent invention (Lynch et al, 1992, Proc. Natl. Acad. Sci. USA 89,1138-1142).

The present invention also relates to a method for the treatment of ahuman or animal organism, comprising administering to said organism atherapeutically effective amount of the adenoviral particle, theeukaryotic cell or the composition of the invention.

A

therapeutically effective amount

is a dose sufficient for the alleviation of one or more symptomsnormally associated with the disease or condition desired to be treated.When prophylactic use is concerned, this term means a dose sufficient toprevent or to delay the establishment of a disease or condition.

The method of the present invention can be used for preventive purposesand for therapeutic applications relative to the diseases or conditionslisted above. It is to be understood that the present method can becarried out by any of a variety of approaches. For this purpose, theadenoviral particle, the host cell or the composition of the inventioncan be is administered directly in vivo by any conventional andphysiologically acceptable administration route, for example byintratumoral injection or by intravenous administration using specificdelivery means adapted to this administration route. Alternatively, theex vivo approach may also be adopted as described above to the inventioninto cells. Prevention or treatment of a disease or a condition can becarried out using the present method alone or, if desired, inconjunction with presently available methods (e.g. radiation,chemotherapy and/or surgery). For example, the method according theinvention can be improved by combining injection with increase ofpermeability of a vessel. In a particular preferred embodiment, saidincrease is obtained by increasing hydrostatic pressure (i.e. byobstructing outflow and/or inflow), osmotic pressure (i.e. withhypertonic solution) and/or by introducing a biologically activemolecule (i.e. histamine into the administered composition; WO98/58542).Furthermore, in order to improve the transfection rate, the patient mayundergo a macrophage depletion treatment prior to administration of thecomposition of the invention (see for example Van Rooijen et al., 1997,TibTech, 15, 178-184).

As discussed above, the method of the present invention is more intendedfor the treatment of cancers, to provide tumor inhibition growth ortumor regression. For example, tumor inhibition may be determined bymeasuring the actual tumor size over a period of time. Morespecifically, a variety of radiologic imaging methods (e.g., singlephoton and positron emission computerized tomography; see generally,“Nuclear Medicine in Clinical Oncology,” Winkler, C. (ed.)Springer-Verlag, New York, 1986), may be utilized to estimate tumorsize. Such methods may also utilize a variety of imaging agents,including for example, conventional imaging agents (e.g., Gallium-67citrate), as well as specialized reagents for metabolite imaging,receptor imaging, or immunologic imaging (e.g., radiolabeled monoclonalantibody specific tumor markers). In addition, non-radioactive methodssuch as ultrasound (see, “Ultrasonic Differential Diagnosis of Tumors”,Kossoff and Fukuda, (eds.), Igaku-Shoin, New York, 1984), may also beutilized to estimate the size of a tumor.

In addition to the in vivo methods for determining tumor inhibitiondiscussed above, a variety of in vivo methods may be utilized in orderto predict in vivo tumor inhibition. Representative examples includelymphocyte mediated anti-tumor cytolytic activity determined forexample, by a ⁵¹Cr release assay, tumor dependent lymphocyteproliferation (Ioannides et al., 1991, J. Immunol. 146, 1700-1707), invitro generation of tumor specific antibodies (Herlyn et al., 1984, J.Immunol. Meth. 73, 157-167, cell (e.g., CTL, helper T cell) or humoral(e.g., antibody) mediated inhibition of cell growth in vitro (Gazit etal., 1992, Cancer Immunol. Immunother 35, 135-144), and, for any ofthese assays, determination of cell precursor frequency (Vose, 1982,Int. J. Cancer 30, 135-142).

Alternatively, inhibition of tumor growth may be determined based upon achange in the presence of a tumor marker. Examples include prostatespecific antigen (“PSA”) for the detection of prostate cancer andCarcino-Embryonic Antigen (“CEA”) for the detection of colorectal andcertain breast cancers. For yet other types of cancers such as leukemia,inhibition of tumor growth may be determined based upon the decreasednumbers of leukemic cells in a representative blood cell count.

When the method of the invention uses recombinant adenoviral particleengineered to express a suicide gene, it can be advantageous toadditionally administer a pharmaceutically acceptable quantity of aprodrug which is specific for the expressed suicide gene product. Thetwo administrations can be made simultaneously or consecutively, butpreferably the prodrug is administered after the adenoviral particleinjection. By way of illustration, it is possible to use a dose ofprodrug from 50 to 500 mg/kg/day, a dose of 200 mg/kg/day beingpreferred. The prodrug is administered in accordance with standardpractice. The oral route is preferred. It is possible to administer asingle dose of prodrug or doses which are repeated for a timesufficiently long to enable the toxic metabolite to be produced withinthe host organism or the target cell. As mentioned above, the prodrugganciclovir or acyclovir can be used in combination with the TK HSV-1gene product and 5-FC in combination with the cytosine deaminase and/oruracil phosphotransferase gene product.

Finally, the present invention also relates to the use of a modifiedadenoviral fiber, of a trimer therof, of an adenoviral particle, of acomposition or of an eukaryotic host cell of the invention having theabove-defined characteristics, to substantially reduce the binding to atleast one native glycosaminoglycan and/or sialic acid-containingreceptor, and especially to HSG receptors. Preferably, said modifiedadenoviral fiber, trimer therof, adenoviral particle, composition oreukaryotic host cell has an affinity for said native glycosaminoglycanand/or sialic acid-containing receptor of at least about one order ofmagnitude less as compared to a wild type adenoviral fiber, trimertherof, adenoviral particle, composition or eukaryotic host cell trimer.

In one embodiment, the modified adenoviral fibers, trimer therof,adenoviral particles, compositions or eukaryotic host cells of theinvention are preferably used to substantially reduce or inhibit thebinding to glycosaminoglycan-containing receptors, and especially to HSGreceptors. In this regard, the amino acid residue(s) to be mutated inthe modified fiber of the invention is (are) within about 5 amino acidsof an amino acid corresponding to residues 404-406, 449-454, 505-512,551-560 of the wild-type Ad5 fiber (SEQ ID NO: 1). It is within thescope of those skilled in the art to identify the equivalent positionsof these Ad5 fiber residues in another adenoviral fiber, on the basis ofavailable sequence database (see for example FIG. 9 of Xia et al., 1994,Structure 2, 1259-1270 giving alignment of the fiber knob regions ofAd2, Ad5, Ad3, Ad7, Ad40, Ad41 and CAV or Van Raaij, 1999, Virology262(2), 333). More preferably, the mutation affects one or more aminoacid residue(s) selected from the group of residues consisting of thethreonine in position 404, the alanine in position 406, the valine inposition 452, the lysine in position 506, the histidine in position 508,and the serine in position 555 of the wild type Ad5 fiber protein asshown in SEQ ID NO: 1. Even more preferably, the modified fiber proteinof the invention comprises at least one substitution mutation of aresidue corresponding to residues 404, 406, 452, 506, 508, and/or 555 ofthe wild-type Ad5 fiber (SEQ ID NO: 1). Most preferably, said mutationof the Ad5 fiber comprises:

-   -   the substitution of the threonine in position 404 by a small        aliphatic residue, such as alanine, proline or glycine, with a        special preference for glycine,    -   the substitution of the alanine in position 406 by a basic        residue such as lysine, arginine or histidine, with a special        preference for lysine,    -   the substitution of the valine in position 452 by a basic        residue such as lysine, arginine or histidine, with a special        preference for lysine,    -   the substitution of the lysine in position 506 by a slightly        basic amide residue such as glutamine or asparagine, with a        special preference for glutamine,    -   the substitution of the histidine in position 508 by a basic        residue such as lysine or arginine, with a special preference        for lysine, or    -   the substitution of the serine in position 555 by a basic        residue such as lysine, arginine or histidine, with a special        preference for lysine,    -   Or any combination thereof.

A representative example of such a combination includes the substitutionof the lysine in position 506 by glutamine and the substitution of thehistidine in position 508 by lysine (K506Q/H508K).

The reduction or alteration of the interaction of said modified fiberand the HSG receptors can be validated as described above and inExamples.

In a second embodiment, the modified adenoviral fibers, trimer therof,adenoviral particles, compositions or eukaryotic host cells of theinvention are preferably used to substantially reduce or inhibit thebinding to both glycosaminoglycan-containing receptors, and especiallyto HSG receptors, and CAR receptors. In this regard, the modified fibercombine any of the modification described above or any combinationthereof with those described before in connection with CAR-ablatedmutants. Preferred example include without limitation the use of amodified adenoviral fiber comprising (i) the substitutiton of the serinein position 408 by glutamic acid, the substitutiton of the lysine inposition 506 by glutamine and the substitutiton of the histidine inposition 508 by lysine (S408E/K506Q/H508K) (ii) the substitutiton of thealanine in position 503 by aspartic acid, the substitutiton of thelysine in position 506 by glutamine and the substitutiton of thehistidine in position 508 by lysine (A503D/K506Q/1-1508K), (iii) thesubstitutiton of the serine in position 408 by glutamic acid and thesubstitutiton of the serine in position 555 by lysine (S408E/S555K), or(iv) the substitutiton of the alanine in position 503 by aspartic acidand the substitutiton of the serine in position 555 by lysine(A503D/S555K).

In a third embodiment, the modified adenoviral fibers, trimer therof,adenoviral particles, compositions or eukaryotic host cells of theinvention are preferably used to substantially reduce or inhibit thebinding to sialic acid-containing receptors. In this regard, the aminoacid residue(s) to be mutated in the modified fiber of the invention is(are) within about 5 amino acids of an amino acid corresponding toresidues 404-410 and 491-505 of the wild-type Ad5 fiber (SEQ ID NO: 1).It is within the scope of those skilled in the art to identify theequivalent positions of these Ad5 fiber residues in another adenoviralfiber, on the basis of available sequence database (see for example FIG.9 of Xia et al., 1994, Structure 2, 1259-1270 giving alignment of thefiber knob regions of Ad2, Ad5, Ad3, Ad7, Ad40, Ad41 and CAV or VanRaaij, 1999, Virology 262(2), 333). Unexpectedly, it has been shown inthis invention that adenovirus particles containing an Ad5 fibermodified in position 408 or 503 have a reduced infectivity toward a CAR⁻lung cell line particularly rich ill sialic acids. In accordance withthese data, a preferred use involves modified fibers or any elementcomprising such a fiber with mutation affecting one or more amino acidresidue(s) selected from the group of residues consisting of the serinein position 408 and the alanine in position 503 and most preferably:

-   -   the substitution of the serine in position 408 by glutamic acid        (S408E),    -   the substitution of the alanine in position 503 by aspartic acid        (A503D), or    -   any combination thereof.

In accordance with the present invention, these modified fibers (e.g. inpositions 408 and/or 503) and any elements containing such a fiber arepreferably used to substantially reduce or inhibit the binding to bothsialic acid-containing receptors and CAR receptors.

In a fourth embodiment, the modified adenoviral fibers, trimer therof,adenoviral particles, compositions or eukaryotic host cells of theinvention are preferably used to substantially reduce or inhibit thebinding to (i) glycosaminoglycan-containing receptors, and especially toHSG receptors, (ii) CAR receptors and (iii) sialic acid-containingreceptors. In this regard, the modified fiber combine any of themodification described in connection with HSG-ablated mutants or anycombination thereof with those described before in connection withCAR-ablated mutants and sialic-acid-ablated variant. Preferred examplesinclude without limitation the use of a modified adenoviral fibercomprising (i) the substitutiton of the serine in position 408 byglutamic acid, the substitutiton of the lysine in position 506 byglutamine and the substitutiton of the histidine in position 508 bylysine (S408E/K506Q/H508K) (ii) the substitutiton of the alanine inposition 503 by aspartic acid, the substitutiton of the lysine inposition 506 by glutamine and the substitutiton of the histidine inposition 508 by lysine (A503D/K506Q/H508K), (iii) the substitutiton ofthe serine in position 408 by glutamic acid and the substitutiton of theserine in position 555 by lysine (S408E/S555K), or (iv) thesubstitutiton of the alanine in position 503 by aspartic acid and thesubstitutiton of the serine in position 555 by lysine (A503D/S555K).

The use of such modified fibers according to the invention allows forreduction of the interaction of such a modified fiber with one or morespecific cellular receptor(s) normally mediating adenovirus attachmentand/or internalization into host cells, and thus significantly restrictthe native tropism od such adenovirus particles. Incorporation of aspecific targeting ligand as described above may be advantageous in thiscontext, in order to redirect adenovirus infection to desired targetcells.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims, the inventionmay be practiced in a different way from what is specifically describedherein.

All of the above cited disclosures of patents, publications and databaseentries are specifically incorporated herein by reference in theirentirety to the same extent as if each such individual patent,publication or entry were specifically and individually indicated to beincorporated by reference.

LEGENDS OF FIGURES

FIG. 1 illustrates the effect of soluble heparin on infection of CHOcells with a series of mutant adenoviruses having the indicated fibermutations.

FIG. 2 illustrates competition assays performed on 293 cells infectedwith either wild-type (wt) or various adenovirus having the indicatedfiber mutation(s) in the presence of 10 μg/ml of recombinant solubleknob.

FIG. 3 illustrates competition assays performed on CHO cells infectedwith either wild-type (wt) or various adenovirus having the indicatedfiber mutation(s) pre-incubated with heparin (30 μg/ml, Sigma);

The following examples serve to illustrate the present invention.

EXAMPLES

The constructs described below are prepared according to the generaltechniques of genetic engineering and of molecular cloning, detailed inSambrook et al. (2001, Molecular Cloning; A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor N.Y.) or according tothe manufacturer's recommendations when a commercial kit is used. Thecloning steps using bacterial plasmids are preferably carried out in theE. coli strain 5K (Hubacek and Glover, 1970, J. Mol. Biol. 50, 111-127)or in E. coli strain BJ5183 (Hanahan, 1983, J. Mol. Biol. 166, 557-580).The latter strain is preferably used for homologous recombination steps.The NM522 strain (Stratagene) is suitable for propagating the M13 phagevectors. The PCR amplification techniques are known to those skilled inthe art (see for example PCR Protocols—A guide to methods andapplications, 1990; Ed Innis, Gelfand, Sninsky and White, Academic PressInc). With respect to the repair of restriction sites, the techniqueused consists in filling the overhanging 5′ ends using the largefragment of E. coli DNA polymerase 1 (Klenow). The Ad5 nucleotidesequences are those disclosed in the Genebank database, under thereference M73260.

With regard to the cell biology, the cells are transfected according tostandard techniques known to those skilled in the art. Mention may bemade of the calcium phosphate precipitation technique, but any otherprotocol can also be used, such as the DEAE dextran technique,electroporation, methods based on osmotic shocks, or methods based onthe use of cationic lipids. In the examples which follow, use is made ofthe human embryonic kidney 293 cell line (ATCC CRL1573), the CHO cellline (ATCC; CCL-61) and 293-Fiber cells (293-Fb), which constitutivelyexpress the adenovirus type 5 fiber protein (described previously inLegrand et al., 1999, J. Virol. 73, 907-919). The culturing conditionsare conventional in the art. For illustrative purposes, the cells aregrown at 37° C. in DMEM (Gibco) supplemented with 10% Fetal Calf Serumand antibiotics.

Materials and Methods

Construction of Fiber-Modified Viral Genomes

All cloning steps were performed using standard molecular biologytechniques. In order to introduce mutations in Ad5 fiber knob, amutagenesis template for the Sculptor™ in vitro mutagenesis system(Amersham, Les Ulis, France) was first generated. The templatesingle-strand DNA m13F5knob contains Ad5 sequence from nucleotide 31994(HindIII site) to nt 32991 (SmaI site) (Santis et al., 1999, J. Gen.Virol. 80, 1519-1527) The substitution mutations were introduced withthe following antisense oligonucleotides: CAR minus Ser408Glu: 5′- gcatt tag tct aca gtt agg ctc tgg agc tgg tgt ggt cca c-3′; (OTG12499; SEQID NO: 2) Ala494Asp (A494D): 5′-gttaggcataaatccaacgtcgtttgtataggctgtgcc-3′; (OTG12728; SEQ ID NO: 3)Ala503Asp (A503D): 5′- accgtgagattttggatagtctgataggttaggcataaa-3′;(OTG12737; SEQ ID NO: 4) Heparan minus Thr404Gly (T404G):5′-ctacagttaggagatggagcgggcccggtccacaaagttagcttatc-3′ (OTG12740; SEQ IDNO: 5) Ala406Lys (A406K): 5′-gtctacagttaggagatggctttggtgtggtccacaaag-3′; (OTG12498; SEQ ID NO: 6)Val452Lys (V452K): 5′- aagatgagcactttgctttgttccagatattgg-3′; (OTG12500;SEQ ID NO: 7) Ser555Lys (S555K): 5′-gtggccagaccagtcccacttaaatgacatagagtatgc-3′; (OTG12506; SEQ ID NO: 8)

The double mutation Lys506Q/His508Lys (K506QH508K) was introduced withfollowing antisense oligonucleotides:5′-acttttggcagttttacccttagactgtggataagctgataggtt-3′ (OTG12738; SEQ IDNO:9).

Ad vectors deficient for CAR and Heparan sulfate proteoglycan pathwayswere constructed with combination of the single S408E or A494D or A503Dor the double A494D/A503D CAR mutations and above triple heparan sulfatemutations K506QH508K/T404G, or K506QH508K/A406K, or K506QH508K/V452K, orK506QH508K/S555K.

The modified fiber was further modified by incorporation of a ligand (7lysine residues also designated 7K) and a flexible linker at theC-terminal extremity of the fiber. For this purpose, the single strandtemplate (either wild type (wt) or mutated m13F5knob) was mutated usingoligonucleotide OTG7000 (5′-aac gat tct tta gct gcc ggg age aga ggc ggaggc gga ggc gct ggg ttc ttg ggc aat-3′ SEQ ID NO: 10) in order tointroduce a 12 amino acid linker (ProSerAlaSerAlaSerAlaSerAlaProGlySer)and then with OTG12125 (5′-cac aaa cga tct tta ctt ctt ctt ttt tct tctttt tgg atc egg gag cga ggc gga g-3′ SEQ ID NO: 11) to add the 7 lysineresidues.

The HindIII-SmaI fragments isolated from the mutated m13F5knob plasmidswere directly introduced by homologous recombination into theBstBI-restricted pTG4213. This plasmid contains a β-galactosidaseexpressing E1-deleted Ad5 genome in which a unique BstBI site wasintroduced at nucleotide 32940, downstream of the fiber stop codon. Thegeneration of the pTG4213 was as follows: m13F5knob was mutated withOTG7213 (5′-t gaa aaa tga ttc gaa att ttc tgc a-3′ SEQ ID NO: 12) tointroduce an unique BsiBI site (sequence in bold). The isolatedHindIII-SmaI fragment was cloned by homologous recombination in the E.coli BJ5183 in pTG8533, a transfer plasmid bearing an Ad5 segmentextending from nt 21562 to the right-end ITR. Thereafter, the purifiedBstEII fragment (nt 24843-35233) was introduced into the Ad5 genome byhomologous recombination with pTG3602, a plasmid containing the fulllength Ad5 genome (described in Chartier et al., 1996, J. Virol. 70,4805-4810). The replacement in this backbone of the E1 region with theMLP driven-βgalactosidase expression cassette was performed as describedpreviously (Legrand et al., 1999, J. Virol. 73, 907-919).

Virus, Production and Titration

Five μg of the fiber-modified viral genomes were excised from theplasmid backbone by PacI digestion and transfected into 293 or 293-Fbcells. Cells were then recovered 2 weeks post-transfection for furtheranalysis and expansion either in wild type 293 cells or in 293-Fb cells,depending on the required complementation (Legand et al., 1999, J.Virol. 73, 907-919). Primary viral stocks were then amplified oil 293cells. Virus purification, titration and storage were as described(Lusky et al., 1999, J. Virol. 73, 8308). Virus particle concentration(P/ml) was measured by optical density (of one OD₂₆₀ corresponds to1.1×10¹² particles/ml). Infectious titers (Infection Unit (IU)/ml) weredetermined 16 h to 20 h post-infection of 293 cells by staining forβ-galactosidase activity (Janes et al., 1986, EMBO J. 5, 3133). Theintegrity of the viral genome and the presence of the fiber mutationwere verified by analysing viral DNA, extracted using the Hirt method(Gluzinan et al., 1983, J. Virol. 45, 91).

Analysis of the Adenoviral Protein Profile

2×10¹⁰ purified viral particles were diluted in 2× Laemmli buffer,incubated for 5 min at 95° C. and loaded onto a 10% SDS-polyacrylamidegel. The proteins were detected by silver staining (Wray et al., 1981,Anal. Biochem. 118, 197). Specific detection of the fiber or penton baseproteins was performed as previously described (Legrand et al., 1999, J.Virol. 73, 907-919).

Competition Experiments

As competitor for CAR entry process, purified Ad5 knob (10 μg/ml) wereused. Target cell monolayer were incubated for one hour at 4° C. witheither PBS or knob molecules. Ad-LacZ bearing either a wild-type or acodified fiber, diluted with 2% FCS-containing DMEM medium, were thenadded to 293 cells for one hour. Cells were then incubated at 37° C. for24 or 48 h pi. After incubation for 24 or 48 hours at 37° C., cells werefixed and stained for beta-galatosidase activity. Alternatively, thebeta-galatosidase activity of whole cell lysate was monitored usingchemiluminescent substrate (luminescent beta-galatosidase detection kit;Clontech, Palo Alto, Calif., USA).

The same technique can be used to evaluate integrin-mediated entryprocess of the different modified fibers, with the exception that a RGDpeptide (4 mg/ml, Neosystem, Strasbourg) was used as a competitor.

As competitor of HSG entry pathway, heparin (3 mg/ml, Sigma, St quentin,France) was used as previously described (Dechecchi et al., 2001, J.Virol. 75, 8772-8780). Wild-type and mutated Ad-LacZ adenoviruses werepreincubated for one hour at 37° C. with heparin (Sigma) at aconcentration of 30 μg/ml in 20 μl of 2% FCS-containing DMEM medium. Thepretreated Adenovirus suspension was then diluted with ice-cold 2%FCS-containing DMEM medium at wanted concentration and added to CHOcells (CAR−) for one hour incubation on ice. Cells were then incubatedat 37° C. for 24 or 48 h pi. Cells were fixed and stained forbeta-galatosidase activity. Alternatively, the beta-galatosidaseactivity of whole cell lysate was monitored using chemiluminescentsubstrate (luminescent beta-galatosidase detection kit; Clontech, PaloAlto, Calif., USA).

Inhibition of adenovirus binding to HSG receptors was performedfollowing heparinase treatment. For this purpose, target cell monolayerwere incubated for one hour at 37° C. with a mix of Heparin Lyase I, II,III (Sigma) at concentration of 100 U/ml. Wild-type or modified Ad-LacZ,diluted with ice-cold 2% FCS-containing DMEM medium at wantedconcentration, were then added to CHO cells for one hour incubation onice. Cells were then incubated at 37° C. for 24 or 48 h pi. Afterincubation for 24 or 48 hours at 37° C., cells were fixed and stainedfor beta-galatosidase activity. Alternatively, the beta-galatosidaseactivity of whole cell lysate was monitored using chemiluminescentsubstrate (luminescent beta-galatosidase detection kit; Clontech, PaloAlto, Calif., USA).

Example 1 Construction of Fiber Mutants Impaired in the HSG EntryPathway and Properties of the HSG-Mutant Viruses

1.1 Incorporation of Modified Fiber in Purified Adenoviral Particles.

Mutations of the Ad5 fiber gene and corresponding adenoviral particleswere generated as described in the Materials and Methods. We sought toinvestigate the fiber residues involved in binding to the HSG receptorswhich have been recently described as cellular receptors for adenovirusinepenidently of CAR. A summary of the fiber mutations altering bindingto HSG receptors is provided in Table 2. TABLE 2 Description of theliber Heparan-mutations Location in the knob Mutations performed AB loop(aa 403-418) Thr404Gly Ala406Lys CD loop (aa 441-453) Val452Lys DG loop(aa 462-514) Lys506Gln/His508Lys I sheet (aa 550-557) Ser555Lys

We first evaluated the effect of these mutation on adenoviral capsidformation. The various fiber-modified viruses produced on 293 cells werepurified by cesium chloride gradient (density 1.34 g/ml). 2×10¹⁰purified particles were subjected to a 4-12% Bis-Tris Nupage gel andtransferred to nitrocellulose. Filters were hybridized with either ananti-penton base serum or an anti-fiber antibody and were then treatedwith a secondary horseradish peroxidase-conjugated donkey anti-rabbitantibody.

The incorporation of the modified fiber into de viral particles wasstudied by Western blot analysis using sera directed against the Ad5Knob (provided by Dr. Gerard; Henry et al., 1994, J. Virol. 68,5239-5246) and the penton base (a polyclonal rabbit anti-penton antibodyprovided by Pr. Boulanger), as control. A strong positive signal wasobserved for the wt Ad-LacZ virus and all the mutated fiber vectors atthe expected molecular weight.

These results demonstrate that the pre-cited mutations have nodeleterious effect on the correct folding of the fiber protein and donot prevent its assembly into the capsid.

1.2 Maturation of Fiber-Modified Viruses

The protein profile of the mutant adenovirus particles bearing amodified fiber as described above was analyzed and compared to Ad-LacZ(having the Ad5 wild-type fiber) and a fiber-deleted controls(Ad-LacZ/Fb^(o)). For this purpose, the various fiber-modified virusesand controls (AdLacZ and Ad-lacZ/Fb^(o)) were produced oil 293 cells andpurified on cesium chloride gradient. 2×10¹⁰ purified particles weresubjected to a 10% SDS-polyacrylamide gel subsequently revealed bysilver staining.

It was found that the majority of the fiber-modified viruses exhibit thesame protein profile as the Ad-LacZ. With the exception of the mutantvirus bearing the A406K fiber, the modified fiber proteins are presentin the viral particle in stoichiometric amounts as the wild-typeadenovirus. On marked contrast, the fiber-deleted Ad-LacZ/Fb^(o) virusstill contains precursors of hexon-associated protein (pVI), of minorcore protein (pVII) and of pVIII protein, indicative of an incompleteproteolytic processing.

1.3 Growth Characteristics of Fiber-Modified Viruses on 293 Cells

Growth properties of the precited fiber-modified Ad were analysed on 293cells. For this purpose, 293 cells were infected at an MOI of 1 IU/cellwith the control viruses Ad-LacZ or Ad-LacZ/Fb^(o) or with the variousfiber-modified viruses. Infected cells and supernatant were harvested at24, 48, 56, 64 and 72 h post-infection and were treated by threefreeze-thawing cycles to release virus particles. Titers of releasedviruses were determined by beta-galactosidase staining.

As a result, the propagation of the fiber-modified viruses is notsignificantly altered as compared to the wild-type adenovirus Ad-LacZ.Consistent with this observation, the titers of infectious mutant virus(IU/ml) after large-scale production was not markedly reduced comparedto the titer obtained with adenovirus bearing a wild type fiber, as wellas the p/IU ratio. This is the consequence of the ability of the mutantadenovirus bearing HSG-ablated fiber to entry 293 cells via CARreceptor. In marked contrast, propagation of CAR-ablated viruses (asdescribed in Leissner et al., 2001, Gene Ther. 8, 49-57) is greatlyaltered in 293 cells as well as that of fiber deleted mutantsAd-LacZ/Fb^(o), as evidenced by the poor formation of infectious units(large augmentation of the IU/perticle ratio).

A summary of these results is provided in Table 3 TABLE 3 Physicalcharacteristics of the fiber mutant viruses Infectious Unit/ml virusParticle/ml (293 cells) IU/Particle Ad-LacZ 2.5 × 10¹²  1.3 × 10¹¹ 1/40Ad-LacZ/Fb° 4.9 × 10¹² 1.1 × 10⁷   1/400000 Ad-LacZ/Fb-Thr404gly 3.1 ×10¹² 2.6 × 10⁸   1/12000 Ad-LacZ/Fb-Ala406Lys 2.2 × 10¹² 2.9 × 10⁸ 1/8000 Ad-LacZ/Fb-Val452Lys 8.8 × 10¹¹ 1.8 × 10⁹  1/500 Ad-LacZ/Fb- 1.2× 10¹² 9.1 × 10⁸  1/1300 Lys506Q/His508Lys Ad-LacZ/Fb-Ser555Lys 1.4 ×10¹²    4 × 10¹⁰ 1/401.4 Ability of Fiber-Modified Ad Virus to Infect CHO Cells in thePresence of Saturating Concentration of Soluble Heparin.

A series of fiber-mutated adenoviruses (including those of Table 2) wereevaluated for their ability to bind cellular HSG receptors by acompetition assay using soluble heparin. One hundred IU/10⁵ cells ofAd-LacZ (equiped with a wild type fiber; corresponding to 4×10³Particles (P)/10⁵ cells), fiberless Ad-LacZ/Fb^(o) (corresponding to4×10⁷P/10⁵ cells) or a series of fiber-modified Ad expressing LacZ.(corresponding to 5×10⁴ to 3×10⁷P/10⁵ cells) were pre-incubated withheparin (30 μg/ml, Sigma) and then added to CHO cells. 48 hpost-infection, the cells were stained for β-galactosidase expression.The efficiency of infection was expressed as the percentage ofβ-galactosidase positive cells in the absence of heparin. The number ofblue cells counted in the control wells (in the absence of knob) rangesfrom 100 to 400.

As shown in FIG. 1, infection of CHO cells (CAR−) by the majority of themutant viruses was greatly reduced in the presence of heparin.Interestingly, five mutant viruses, respectively T404G, A406K, V452K,K506Q/H508K and S555K, were identified which were able to infect CHOcells in the presence of high concentrations of soluble heparin,suggesting that the putative binding of the corresponding viruses to theheparan sulfate was impaired. In marked contrast, for all other mutantadenoviruses including those ablated for CAR binding (e.g. S408E, A494D,A503D, etc.), the infection of the target cells still seemed to bemediated through heparan sulfate suggesting that the corresponding fibermutations have no significant adverse effects on this pathway. Sameresults were obtained with increasing concentration of heparin (10, 30,50 and 100 μg/ml), then suporting specificity of these results. Themutations of the so called Heparan-ablated adenoviruses are located inthe AB loop (T404G; A406K), in the CD loop (V452K), in the DG loop(K506Q/H508K), and in the I sheet (S555K).

1.5 Properties of Heparan Mutants Concerning CAR Pathway

The five Heparan-ablated adenoviruses, bearing respectively T404G,A406K, V452K, K506Q/H508K and S555K fiber mutations, were then testedfor their ability to infect 293 cells. 5×10⁴ 293 cells were infectedwith Ad-LacZ, Ad-LacZ/Fb^(o) as controls or with these fivefiber-modified mutant viruses at a MOI of 1 IU/cell. At 48 hpostinfection, the cells were stained for beta-galactosidase expression.

As a result, the five Heparan-abalted adenovirus still efficientlyinfects 293 cells through CAR binding, with similar infectivity thanthat of wild type adenovirus.

Infection was also performed in the presence of recombinant Ad5 knobprotein as a competitor of CAR binding, For this purpose, 293 cells wereincubated for 30 min at 37° C. with 10 μg/ml of Ad5 knob proteinpurified from a recombinant E. coli strain. One hundred IU/10⁵ cells ofAd-LacZ (corresponding to, 4×10³ P/10⁵ cells), fiberless Ad-LacZ/Fb^(o)(corresponding to 4×10⁷P/10⁵ cells) or Heparan-ablated mutantsexpressing LacZ (T404G, A406K V452K, K506Q/H508K and S555K correspondingto 5×10⁴-3×10⁷ P/10⁵ cells), were then added. 24 h post-infection, thecells were stained for β-galactosidase expression. The efficiency ofinfection was expressed as the percentage of β-galactosidase positivecells in the absence of knob. The number of blue cells counted in thecontrol wells (in the absence of knob) ranges from 100 to 400.

It was shown that the five Heparan-ablated mutant adenovirus are fullycompetited (between 80 and 90% of inhibition) by preincubating the cellswith a saturating concentration of recombinant Ad5 knob, confirming thattheir corresponding fiber mutations do not impair and interfer with CARbinding.

Example 2 Possibility of Retargeting Infection of the Heparan-AblatedMutant Virus By Addition of a Polylysine Ligand at the C-Terminus of theFiber

A polysine ligand was inserted at the C-terminus of the modified fiberablated for HSG binding (T404G, A406K, V452K, K506Q/H508K and S555Kmutations respectively) and LacZ-expressing adenoviral particlesharboring the 7K retargeted and HSG-ablated fiber were constructed asdescribed in the “Materials and Methods” section. The 7K ligand iscomposed of seven lysine residues (7K) and is known to confer theability to efficiently bind heparan sulfate proteoglycans on the surfaceof target cells. In other terms, addition of the 7K ligand to the HSGablated fibers will therefore restore HSG binding, and thus, demonstratethe possibility of retargeting adenovirus tropism as desired (byselecting an appropriate ligand).

As a result, 7K-containing mutant viruses have similar properties astheir mutant conterparts (devoid of ligand), in terms of growthkinetics, maturation, and yield production. However, their ability toinfect cells via heparan sulfate was restored and moreover amplified.These results show that the above HSG-ablated modified fiber canincorporate ligand moieties at the C-terminal extremity of the knob, totarget adenovirus infection to desired cell types.

Example 3 Constructions of Fiber Mutants Impaired in Both HSG and CARPathways of Infection and Properties of These Combinated-Mutant Viruses

Fiber residues involved both in binding to the CAR and HSG receptorswere mutated to provide a modified fiber impaired in both pathways. Inthe context of this invention, any mutation fiber mutations altering CARbinding (e.g. S408E, A503D, A494D) can be combined to any mutationinvolved in HSG binding (e.g. T404G, A406K, V452K, H506Q, H508K, S555K,H506Q/H508K). The following combinations of mutations altering bindingto CAR and HSG receptors and corresponding adenoviral particles weregenerated as described in the Materials and Methods:

Ser408Glu and Val452Lys (S408E/V452K);

Ser408Glu and Lys506Gln/His508Lys (S408E/K506Q/F508K);

Ser408Glu and Ser555Lys (S408E/S555K);

Ala503Asp and Val452Lys (A503D/V452K);

Ala503Asp Glu and Lys506Gln/His508Lys (A503D/K506Q/H508K); and

Ala503Asp and Ser555Lys (A503D/S555K).

3.1 Incorporation of Modified Fiber in Purified Adenoviral Particles

The effect of the combination of CAR⁻ and HSG⁻ mutations on adenoviralcapsid formation was evaluated by Western blotting as described inExample 1 and compared to the incorporation of the wild type Ad5 fiber(Ad-LacZ virus having a wild type fiber as positive control). Thevarious viruses were produced on 293 cells and purified by cesiumchloride gradient (density 1.34 g/ml). 2×10¹⁰ purified particles weresubjected to a 4-12% Bis-Tris Nupage gel and transferred tonitrocellulose. Filters were hybridized either with sera directedagainst the Ad5 Knob (provided by Dr. Gerard; Henry et al., 1994, J.Virol. 68, 5239-5246) or with an anti-penton base serum (polyclonalrabbit anti-penton antibody; provided by Pr. Boulanger), and were thentreated with a secondary horseradish peroxidase-conjugated donkeyanti-rabbit antibody.

A strong positive signal having the expected molecular weight washighlighted for the wt Ad-LacZ virus (positive control) and all themutated fiber vectors.

These results demonstrate that the combinations of CAR⁻ and HSG⁻mutations have no deleterious effect on the correct folding of the fiberprotein and do not prevent its assembly into the capsid.

3.2 Maturation of Combinated Fiber-Modified Viruses

The protein profile of the CAR⁻ and HSG⁻ mutant adenovirus particles wasanalyzed and compared to CAR⁻ mutant Ad-LacZ/Fb S408E (which fiber hasthe mutation Ser408Glu), Ad-LacZ (having the Ad5 wild-type fiber) aspositive control, and Ad-LacZ/Fb^(o) (a fiber-deleted Ad5) as negativecontrol. For this purpose, the various viruses were produced on 293cells and purified on cesium chloride gradient. 2×10¹⁰ purifiedparticles were subjected to a 10% SDS-polyacrylamide gel subsequentlyrevealed by silver staining.

The combined CAR⁻ and HSG⁻ mutant viruses exhibit the same proteinprofile as the Ad-LacZ control and Ad-LacZ/Fb S408E CAR⁻ mutant. In allcases, the modified fiber proteins are present in the viral particle instoichiometric amounts as the wild-type adenovirus. On marked contrast,the fiber-deleted Ad-LacZ/Fb^(o) virus still contains precursors ofhexon-associated protein (pVI), of minor core protein (pVII) and ofpVIII protein, indicative of an incomplete proteolytic processing.

3.3 Growth Characteristics of Fiber-Modified Viruses

Growth properties of the CAR⁻ and HSG⁻ mutant adenoviruses were analysedon 293 cells and compared to the growth of single modified counterpartsCAR⁻ mutant Ad-LacZ/Fb S408E, HSG⁻ mutant viruses (Ad-LacZ/Fb V452K,Ad-LacZ/Fb K506Q/H508K, or Ad-LacZ/Fb S555K), and Ad-LacZ positivecontrol. 293 cells were infected at a MOI of 10 particles/cell with thevarious mutant viruses or controls. Infected cells and supernatant wereharvested at 24, 48, 56, 64 and 72 h post-infection and were treated bythree freeze-thawing cycles to release virus particles. Titers ofreleased viruses were determined by beta-galactosidase staining.

The propagation of CAR⁻ and HSG⁻ ablated mutant viruses is greatlyaltered in 293 (CAR+) cells, as evidenced by the poor formation ofinfectious units even 721 h post-infection. It should be noted that thea poor virus yield is also obtained with the CAR⁻ Ad-LacZ/Fb S408E. Inmarked contrast, propagation of HSG⁻ mutant viruses Ad-LacZ/Fb V452K,Ad-LacZ/Fb K506Q/H508K, and Ad-LacZ/Fb S555K is efficient and in thesame range of that obtained with the wild-type adenovirus Ad-LacZ givingbetween 8 to 10×10E8 p/ml at 72 h post-infection. These resultscorrelate with the CAR⁻ phenotype of the impaired mutants.

After large-scale production, the titers of infectious mutant virus(IU/ml) was also markedly reduced compared to the titer obtained withadenovirus bearing a wild type fiber. This correlates with the largeaugmentation of the IU/particle ratio, as illustrated in Table 4. TABLE4 Physical characteristics of the fiber mutant viruses Particle/mlInfectious Unit (IU)/ virus (P) ml (293 cells) IU/Particle Ad-LacZ 2.5 ×10¹²  1.3 × 10¹¹ 1/40   Ad-LacZ/Fb-S408E 8.6 × 10¹² 8.2 × 10⁸ 1/10487Ad-LacZ/Fb- 1.15 × 10¹²  1.1 × 10⁸ 1/10454 S408E/V452K Ad-LacZ/Fb- 1.88× 10¹²  4.7 × 10⁸ 1/4000  S408E/K506Q/H508K Ad-LacZ/Fb- 2.1 × 10¹² 1.34× 10⁸  1/15671 S408E/S555K

These results are the consequence of the fiber mutations affectingresidues involved in CAR and HSG binding, thus hampering the combinedCAR⁻ and HSG⁻ mutant adenovirus to entry 293 cells via CAR receptor.

3.4 Properties of Combined Mutant Viruses Concerning CAR Pathway

The infectivity of CAR+293 cells of the combined CAR⁻ and HSG-ablatedadenoviruses was evaluated in the presence or in the absence of knobcompetitor. 5×10⁴ 293 cells were infected with either CAR⁻ andHSG⁻-ablated adenoviruses (Ad-LacZ/Fb-S408E/N452K,Ad-LacZ/Fb-S408E/K506Q/0508K and Ad-LacZ/FbS408E/S555K, respectively) orAd-LacZ and Ad-LacZ/FbS408E as controls at a MOI of 100 particles/cell.At 24 h post-infection, the cells were stained for beta-galactosidaseexpression. As mentioned in section 3.3, all CAR⁻ and HSG⁻ mutants donot efficiently infect 293 cells due to thir impairement in CAR binding,similarly to Ad-LacZ/Fb S408E mutant.

Infection was also performed in the presence of recombinant Ad5 knobprotein as a competitor of CAR binding. For this purpose, 293 cells wereincubated for 30 min at 37° C. with 10 μg/ml of Ad5 knob proteinpurified from a recombinant E. coli strain before being infected withone hundred particles/cells of Ad-LacZ, CAR-deficient Ad-LacZ/S408E orcombined CAR⁻ and HSG⁻ablated mutants expressing LacZ (8408E/V452K;S408E/K506Q/H508K; and S408E/S555K, respectively). 24 h post-infection,the cells were stained for beta-galactosidase expression. The efficiencyof infection was expressed as the percentage of beta-galactosidasepositive cells in the absence of knob. The number of blue cells countedin the control wells (in the absence of knob) ranges from 100 to 400.

As illustrated in FIG. 2, 293 infection of Ad-LacZ is stronglycompetited by soluble knob (approximately 90% inhibition) as expecteddue to the blockage of the CAR pathway by the recombinant knob. Asdescribed in Example 1, the HSG− mutants (V452K, K506Q/H508K and S555K)are also strongly competited by preincubating the 293 cells with asaturing concentration of recombinant knob. In marked contrast, thecombined CAR⁻ and HSG⁻ ablated mutant adenovirus are not or very poorlycompetited (between 0 and 10% of inhibition) by recombinant Ad5 knob, aswell as the CAR⁻ deficient fiber S408E virus control, confirming theirunability to use CAR pathway. These results show that the HSG⁻ mutations(V452K, K506Q/H508K and S555K) do not interfer with the S408E mutationablating binding to CAR.

3.5 Properties of Combined Mutant Viruses Concerning HSG Pathway

The ability of CAR⁻ and HSG⁻ mutant adenoviruses to bind cellular HSGreceptors was evaluated by a competition assay using soluble heparin.CAR deficient CHO cells were infected at a MOI of five hundredparticles/cells by either the combined CAR⁻ and HSG⁻ mutant adenovirusor control viruses, the CAR⁻ Ad-LacZ/Fb S408E mutant, the HSG⁻ virusmutants (Ad-LacZ/Fb V452K, Ad-LacZ/Fb K506Q/H508K, and Ad-LacZ/Fb S555Krespectively), or the positive control Ad-LacZ (equiped with a wild typefiber). Competition assays were performed by pre-incubating the viruswith heparin (30 μg/ml, Sigma) before infecting CHO cells. 48 hpost-infection, the cells were stained for beta-galactosidaseexpression. The efficiency of infection was expresses as the percentageof beta-galactosidase positive cells in the absence of heparin. Thenumber of blue cells counted in the control wells (in the absence ofknob) ranges from 100 to 400.

As shown in FIG. 3, infection of CHO cells (CAR−) by Ad-LacZ and Ad/FbS408E was greatly reduced in the presence of heparin, indicating thatthe infection of the target cells still seemed to be mediated throughheparan. In contrast, the combined mutant viruses, Ad-LacZ (S408E/V452K,S408E/K506Q/H508K and S⁴08E/S555K, respectively) were able to infect CHOcells in the presence of high concentrations of soluble heparin with thesame efficiency than in the absence of heparin, suggesting that theputative binding of the corresponding viruses to CHO cells isindependent of the heparan sulfate proteoglycans. Same results wereobtained with increasing concentration of heparin (10, 30, 50 and 100μg/ml), then suporting specificity of these results.

These experiments confirm that the combination with CAR⁻ mutation(S408E) do not interfer with the HSG ablating binding V452K, K506Q/H508Kand S555K mutations.

The combination of HSG mutation (V452K, K506Q/H508K and S555K) with theCAR⁻ mutation (A503D) can be tested as illustrated in Example 3.

1. A modified adenoviral fiber containing at least one mutation affecting one or more amino acid residue(s) of said adenoviral fiber interacting with at least one glycosaminoglycan and/or sialic acid-containing cellular receptor.
 2. The modified adenoviral fiber according to claim 1, wherein said modified adenoviral fiber has an affinity for said glycosaminoglycan or sialic acid-containing cellular receptor of at least about one order of magnitude less than a wild-type adenoviral fiber.
 3. The modified adenoviral fiber according to claim 1, wherein said glycosaminoglycan-containing cellular receptor is a heparin- or heparan sulfate-containing cellular receptor.
 4. The modified adenoviral fiber according to claim 3, wherein said heparin- or heparan sulfate-containing cellular receptor is a heparan sulfate glycosaminoglycan (HSG) cellular receptor which normally interacts with the wild-type adenoviral fiber to mediate adenovirus attachment to a host cell.
 5. The modified adenoviral fiber according to claim 1, wherein said mutation affects one or more amino acid residue(s) within the AB loop, the CD loop, the DG loop and/or the beta sheet I of the knob.
 6. The modified adenoviral fiber according to claim 1, wherein said mutation affects one or more amino acid residue(s) selected from the group of residues consisting of the threonine in position 404, the alanine in position 406, the valine in position 452, the lysine in position 506, the histidine in position 508, and the serine in position 555 of the wild type Ad5 fiber protein as shown in SEQ ID NO:
 1. 7. The modified adenoviral fiber according to claim 6, wherein said mutation comprises: The substitution of the threonine in position 404 by glycine, The substitution of the alanine in position 406 by lysine, The substitution of the valine in position 452 by lysine, The substitution of the lysine in position 506 by glutamine, The substitution of the histidine in position 508 by lysine, or The substitution of the serine in position 555 by lysine, Or any combination thereof.
 8. The modified adenoviral fiber according to claim 6, wherein said mutation comprises: the substitution of the lysine in position 506 by glutamine and the substitution of the histidine in position 508 by lysine; the substitution of the threonine in position 404 by glycine, the substitution of the lysine in position 506 by glutamine and the substitution of the histidine in position 508 by lysine; the substitution of the alanine in position 406 by lysine, the substitution of the lysine in position 506 by glutamine and the substitution of the histidine in position 508 by lysine; the substitution of the valine in position 452 by lysine, the substitution of the lysine in position 506 by glutamine and the substitution of the histidine in position 508 by lysine; the substitution of the lysine in position 506 by glutamine, the substitution of the histidine in position 508 by lysine and the substitution of the serine in position 555 by lysine.
 9. The modified adenoviral fiber according to claim 1, wherein said mutation affects one or more amino acid residue(s) selected from the group of residues consisting of the threonine in position 404, the aspartic acid in position 406, the valine in position 452, the lysine in position 506, the glutamine in position 508, and the threonine in position 556 of the wild type Ad2 fiber protein.
 10. The modified adenoviral fiber according to claim 1, wherein said modified adenoviral fiber further comprises at least one additional mutation affecting one or more amino acid residue(s) of said adenoviral fiber interacting with the CAR cellular receptor.
 11. The modified adenoviral fiber according to claim 10, wherein said modified adenoviral fiber has an affinity for said CAR cellular receptor and said glycosaminoglycan and/or sialic acid-containing cellular receptor of at least about one order of magnitude less than a wild-type adenoviral fiber.
 12. The modified adenoviral fiber according to claim 10, wherein said additional mutation affects one or more amino acid residue(s) selected from the group consisting of the serine in position 408, the proline in position 409, the arginine in position 412, the lysine in position 417, the lysine in position 420, the tyrosine in position 477, the arginine in position 481, the leucine in position 485, the tyrosine in position 491, the alanine in position 494, the phenylalanine in position 497, the methionine in position 498, the proline in position 499 and the alanine in position 503 of the wild type Ad5 fiber protein as shown in SEQ ID NO:
 1. 13. The modified adenoviral fiber according to claim 12, wherein said additional mutation comprises: the substitution of the serine in position 408 by glutamic acid (S408E), the substitution of the proline in position 409 by lysine (P409K), the substitution of the tyrosine in position 477 by alanine (Y477A), the substitution of the leucine in position 485 by lysine (L485K), the substitution of the tyrosine in position 491 by aspartic acid (Y491D), the substitution of the alanine in position 494 by aspartic acid (A494D), the substitution of the phenylalanine in position 497 by aspartic acid (F497D), the substitution of the methionine in position 498 by aspartic acid (M498D), the substitution of the proline in position 499 by glycine (P499G), the substitution of the alanine in position 503 by aspartic acid (A503D), or any combination thereof.
 14. The modified adenoviral fiber according to claim 13, wherein said modified adenoviral fiber comprises (i) the substitution of the serine in position 408 by glutamic acid, the substitution of the lysine in position 506 by glutamine and the substitution of the histidine in position 508 by lysine (S408E/K506Q/H508K) (ii) the substitutiton of the alanine in position 503 by aspartic acid, the substitutiton of the lysine in position 506 by glutamine and the substitutiton of the histidine in position 508 by lysine (A503D/K506Q/H508K), (iii) the substitutiton of the serine in position 408 by glutamic acid and the substitutiton of the serine in position 555 by lysine (S408E/S555K), or (iv) the substitutiton of the alanine in position 503 by aspartic acid and the substitutiton of the serine in position 555 by lysine (A503D/S555K).
 15. The modified adenoviral fiber according to claim 1, wherein said modified adenoviral fiber trimerizes when produced in a eukaryotic host cell.
 16. A trimer comprising the modified adenoviral protein of claim
 1. 17. The trimer according to claim 16, having an affinity for a native glycosaminoglycan and/or sialic acid-containing receptor of at least about one order of magnitude less than a wild type adenoviral fiber trimer.
 18. The trimer according to claim 16, containing a modified adenoviral fiber, wherein said trimer further has an affinity for a native CAR cellular receptor of at least about one order of magnitude less than a wild type adenoviral fiber trimer.
 19. A DNA fragment or expression vector encoding the modified adenoviral fiber of claim
 1. 20. An adenoviral particle lacking a wild-type fiber and comprising the trimer of claim
 16. 21. The adenoviral particle of claim 20, further comprising one or more penton base having a mutation affecting at least one native RGD sequence.
 22. The adenoviral particle of claim 20, further comprising a ligand.
 23. The adenoviral particle of claim 22, wherein said ligand binds at least one cell-surface anti-ligand other than a native receptor which normally mediates cell attachment and/or uptake of a wild-type adenovirus.
 24. The adenoviral particle of claim 23, wherein said cell surface anti-ligand is selected from the group consisting of cell-specific markers, tissue-specific receptors cellular receptors, antigenic peptides, tumor-associated markers, tumor-specific receptors and disease-specific antigens.
 25. The adenoviral particle of claim 22, wherein said ligand is immunologically, chemically or genetically coupled to a viral polypeptide exposed at the surface of said adenoviral particle.
 26. The adenoviral particle of claim 25, wherein said viral polypeptide exposed at the surface of said adenoviral particle is selected from the group consisting of penton base, hexon, fiber, protein IX, protein VI and protein IIIa.
 27. The adenoviral particle of claim 26, wherein said ligand is genetically inserted in said modified fiber, especially at the C-terminus or within the HI loop.
 28. The adenoviral particle of claim 26, wherein said ligand is genetically inserted in the protein pIX, especially at the C-terminus or within the C-terminal portion of said protein pIX.
 29. The adenoviral particle of claim 20, which is an empty capsid.
 30. The adenoviral particle of claim 20, comprising an adenoviral genome.
 31. The adenoviral particle of claim 30, wherein said adenoviral genome is replication-defective.
 32. The adenoviral particle of claim 30, wherein said adenoviral genome comprises at least one gene of interest placed under the control of the regulatory elements allowing its expression in a host cell.
 33. The adenoviral particle of claim 32, wherein said regulatory elements allowing the expression of said gene of interest are functional within a host cell presenting at its surface an anti-ligand to which said ligand binds.
 34. The adenoviral particle of claim 32, wherein said regulatory elements comprise a promoter selected from the group consisting of tissue-specific promoters and tumor-specific promoters.
 35. A process for producing the adenoviral particle according to claim 20, comprising the steps of: Introducing said adenoviral particle or the genome of said adenoviral particle into a suitable cell line, Culturing said cell line under suitable conditions so as to allow the production of said adenoviral particle, and Recovering the produced adenoviral particle from the culture of said cell line, and Optionally purifying said recovered adenoviral particles.
 36. The process according to claim 35, wherein said adenoviral particle is replication-defective and said cell line complements at least one defective function of said adenoviral particle.
 37. The process of claim 35, wherein said cell line comprises either in a form integrated into the genome or in episome form a DNA fragment or an expression vector encoding the modified adenoviral fiber containing at least one mutation affecting one or more amino acid residue(s) of said adenoviral fiber interacting with at least one glycosaminoglycan and/or sialic acid-containing cellular receptor.
 38. The process according to claim 37, wherein said cell line is further capable of complementing one or more adenoviral functions selected from the group consisting of the functions encoded by the E1, E2, E4, L₁, L2, L3, L4, L5 regions or any combination thereof.
 39. The process according to claim 37, wherein said cell line is produced from the 293 cell line or from the PER C6 cell line.
 40. A composition comprising the adenovirus particle according to claim 20, in combination with a vehicle which is acceptable from a pharmaceutical point of view.
 41. The composition of claim 40, wherein said adenovirus particle is conjugated to a lipid or polymer.
 42. A method for the preparation of a drug intended for the treatment or the prevention of a disease in a human or animal organism by gene therapy comprising using the adenovirus particle according to claim
 20. 43. The method according to claim 42, wherein the disease is a cancer, including glioblastoma, sarcoma, melanomas, mastocytoma, carcinomas as well as breast, prostate, testicular, ovarian, cervix, lung, kidney, bladder, liver, colon, rectum, pancreas, stomac, esophagus, larynx, brain, throat, skin, central nervous system, blood, and bone cancers. 